From the Department of Biochemistry, University of
Nebraska, Lincoln, Nebraska 68588, the § SmithKline Beecham
Pharmaceuticals, King of Prussia, Pennsylvania 19406, the
¶ Laboratory of Experimental Carcinogenesis, NCI, National
Institutes of Health, Bethesda, Maryland 20892, and the
Basic
Research Laboratory, NCI, National Institutes of Health,
Bethesda, Maryland 20892
Received for publication, June 1, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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Animal thioredoxin reductases (TRs) are
selenocysteine-containing flavoenzymes that utilize NADPH for reduction
of thioredoxins and other protein and nonprotein substrates. Three
types of mammalian TRs are known, with TR1 being a cytosolic enzyme,
and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3
occurred as homodimers of 55-57-kDa subunits. We report here that TR1
isolated from mouse liver, mouse liver tumor, and a human T-cell line
exhibited extensive heterogeneity as detected by electrophoretic,
immunoblot, and mass spectrometry analyses. In particular, a 67-kDa
band of TR1 was detected. Furthermore, a novel form of mouse TR1
cDNA encoding a 67-kDa selenoprotein subunit with an additional
N-terminal sequence was identified. Subsequent homology analyses
revealed three distinct isoforms of mouse and rat TR1 mRNA. These
forms differed in 5' sequences that resulted from the alternative use
of the first three exons but had common downstream sequences.
Similarly, expression of multiple mRNA forms was observed for human
TR3 and Drosophila TR. In these genes, alternative first
exon splicing resulted in the formation of predicted mitochondrial and
cytosolic proteins. In addition, a human TR3 gene overlapped with the
gene for catechol-O-methyltransferase (COMT) on a
complementary DNA strand, such that mitochondrial TR3 and
membrane-bound COMT mRNAs had common first exon sequences; however,
transcription start sites for predicted cytosolic TR3 and soluble COMT
forms were separated by ~30 kilobases. Thus, this study demonstrates
a remarkable heterogeneity within TRs, which, at least in part, results
from evolutionary conserved genetic mechanisms employing alternative
first exon splicing. Multiple transcription start sites within TR genes
may be relevant to complex regulation of expression and/or organelle-
and cell type-specific location of animal thioredoxin reductases.
The thioredoxin redox system is one of two major redox systems in
animal cells, which, together with the glutathione system, participates
in the redox control of a great variety of biological processes
involved in cell life and death (1-3). Disruption of the gene for
thioredoxin 1, a 12-kDa thiol disulfide oxidoreductase, results in
embryonic lethality in mice (4) demonstrating an essential role of the
thioredoxin system in the development of mammals. Three distinct
thioredoxin reductases
(TRs)1 (5-10) are
responsible for maintaining thioredoxins in a reduced state and are
also capable of reducing a great variety of other protein and
nonprotein redox substrates. The cytosolic thioredoxin reductase (TR1),
the most characterized of the three enzymes, was known for decades, but
only recently it was found to be a selenium-containing protein (11).
TR1 contains a C-terminal penultimate selenocysteine residue encoded by
TGA (12), and this residue is essential for catalytic activity of the
enzyme (13-15).
One of the first indications that multiple TR forms occur in mammalian
cells was immunoblot analyses of various human TR preparations. An
enzyme possessing TR activity that was isolated from a human lung
adenocarcinoma cell line NCI-H441 failed to react with antibodies specific for rat liver TR1 (11), whereas TR isolated from a human
T-cell line JPX9 reacted with these antibodies (12). Determination of
internal peptide sequences of the T-cell TR demonstrated that this
protein was TR1 (12). Unfortunately, the NCI-H441 enzyme was not
sequenced, and in retrospect it seems possible, that this TR could have
been encoded by a different gene. In addition to a TR form not reacting
with anti-TR1 antibodies, subsequent studies identified two forms of
TR1 isolated from either NCI-H441 or HeLa cells that showed a positive
immunoblot signal with these antibodies (16-18). Yet, these forms
differed in catalytic activity, selenium content, and affinity for
column matrices. In particular, forms that differed in the ability to
bind to a heparin column were extensively characterized (16-18).
However, differences in either protein or gene sequences, or in
post-translational modifications that were responsible for the observed
changes in catalytic and chromatographic properties, were not reported.
Recently, two additional thioredoxin reductases, TR2 and TR3, were
identified that contained the conserved selenocysteine residue (5-10).
These enzymes had also other sequences essential for catalytic
activity, including an N-terminal disulfide active center, NADPH- and
FAD-binding domains and dimer interface sequences (5-10). The three TR
enzymes showed >50% sequence identity, although TR1 and TR2 were
closely related enzymes, while TR3 was a more evolutionary distant
enzyme (10). TR3 (also called TR In the present report, we describe the occurrence of multiple forms of
TR1 and TR3 and utilize genetic and biochemical techniques to
characterize the basis for heterogeneity within animal TR preparations. We find differences in 5'-end cDNA sequences for mammalian TR1, mammalian TR3, and Drosophila TR that resulted from the
alternative use of first exons.
Partial Isolation of Mouse TRs from Normal Livers and Liver
Tumors on an ADP-Sepharose Column--
Male wild type and transforming
growth factor TR1 Purification--
TR1 was isolated from mouse liver, rat
prostate, and a human T-cell line JPX9 according to a three-step
procedure that involved DEAE-Sepharose (Amersham Pharmacia Biotech),
ADP-Sepharose, and phenyl-HPLC columns (TosoHaas) as described
previously (12). To obtain 75Se-labeled TR1, unlabeled
mouse livers were mixed with a single 75Se-labeled wild
type liver. Isolated proteins were analyzed by immunoblot assays with
antibodies specific for TR1, TR2, and TR3 (10), protein staining with
Coomassie Blue and by detecting 75Se with a PhosphorImager
(Molecular Dynamics). To further fractionate the enzyme, TR1
preparation was dialyzed against 20 mM Tris-HCl, pH 7.0, and applied onto a heparin-HPLC column (TosoHaas). The proteins were
eluted with a gradient of 20-500 mM Tris-HCl, pH 7.0. 75Se was detected in fractions with a Two-dimensional Gel Electrophoresis--
Two-dimensional gel
electrophoresis of 75Se-contining polypeptides was
performed as described previously (20). Briefly, 25 µl of each TR
preparation (500 µg of total protein), which was partially purified
on an ADP-Sepharose column, were resolved in the first dimension on an
isoelectric focusing (IEF) gel, followed by separation in the second
dimension on a 10% polyacrylamide gel under reducing conditions. The
separated polypeptides were electroblotted on polyvinylidene
difluoride membranes, the blots stained with Ponceau S, and the
75Se-containing proteins detected on the dried membranes by
the PhosphorImager analysis.
Peptide Mapping--
TR samples purified by one-dimensional or
two-dimensional gel electrophoresis were transferred onto
polyvinylidene difluoride membranes and stained with either Ponceau S
or Sulforodamine B. Bands were excised and digested with trypsin as
described (21). After digestion, peptides were extracted from the
membrane with the addition of 10 µl of formic acid:ethanol (1:1) for
1 h at room temperature. 0.5 µl were sampled directly from the
supernatant to the matrix-assisted laser desorption ionization
(MALDI) plate, mixed with 0.5 µl of Mass Spectrometric Analysis of Intact Human TR
Molecules--
Mass spectrometric analysis of native or alkylated
human TR proteins was performed on a API300 triple quadrupole mass
spectrometer equipped with a Micro-IonSpray source. Prior to MS
analysis proteins were purified by RP-HPLC on a narrow-bore Vydac C4
column (150 × 2.1 mm, 5 µm) using a linear gradient of 0-70%
acetonitrile containing 0.1% trifluoroacetic acid and a flow rate of
250 µl/min. Samples were manually collected and directly injected
into the mass spectrometer ion source by infusion at 1 µl/min.
Reduction and alkylation of TRs was conducted in denaturing conditions
(6 M guanidine HCl, pH 8.0) using iodoacetamide as an
alkylating agent.
Other Procedures--
Mouse cDNA clones (clone ID 607552, accession number AI662374, Stratagene mouse skin cDNA library; and
clone ID 2064717, accession number AI789478, Sugano mouse kidney-mkia
cDNA library) were purchased from Research Genetics and their
insert sequences experimentally determined. The two clones overlapped
for 510 nucleotides and the combined cDNA sequence was 3626 nucleotides long. SDS-PAGE, immunoblot, and isoelectrofocusing analyses
were performed with electrophoretic supplies and gels from Novex.
Immunoblot membranes were developed with a SuperSignal detection kit
(Pierce) or an ECL detection kit (Amersham Pharmacia Biotech) using
previously developed antibodies specific for TR1, TR2, and TR3 (10). We have previously demonstrated strict isozyme specificity of these antibodies (10). Nucleotide sequences were analyzed with BLAST programs
(23). Exons were predicted with a NetGene2 program. Mitochondrial signal peptides were predicted with the PSORT II or
SignalP programs.
Heterogeneity within Mouse Thioredoxin Reductase
Preparations--
To determine whether mouse TR1 occurs in multiple
forms and if the distribution of these forms differs between normal and malignant cells, we partially isolated TRs from a pool of
75Se-labeled wild type mouse livers and from a pool of
75Se-labeled liver tumors developed in
TGF
When 75Se-labeled protein extracts obtained from either
normal livers or liver tumors were fractionated on an ADP-Sepharose column, TR1, seen as a 75Se-labeled band (Fig.
1A, lanes 3 and 6),
and TR3, which is masked by TR1 due to its lower abundance (6, 10),
were separated from the remainder of 75Se-labeled proteins
(Fig. 1A). Two other major 75Se-labeled proteins
that did not bind to the column were glutathione peroxidase 1 (GPx1)
and glutathione peroxidase 4 (GPx4) (19). Since we used identical
procedures for fractionation of normal and tumor samples, comparison of
GPx1 and other selenoprotein bands in normal (Fig. 1A, lane
1) and tumor (Fig. 1A, lane 4) samples suggested that
the levels of GPx1 were decreased in tumors. A similar decrease was
previously observed when normal livers from TGF
In addition to the mouse samples, we analyzed TR1 isolated from rat
prostate by a standard three-column procedure (Fig. 1B, lane
3), and a TR preparation isolated from a human epidermoid A431
cell line using a single ADP-Sepharose column (Fig. 1B, lane 4). Interestingly, although the major form of human TR1 migrated as a 57-kDa band, human TR3 was present as a mixture of 55-57-kDa species.
While mouse TR preparations isolated from normal and tumor samples
migrated as single species and exhibited similar electrophoretic properties on SDS-PAGE gels (Fig. 1B), this electrophoretic
technique is often insufficient to resolve minor differences within
preparations. Therefore, to further test protein preparations shown in
Fig. 1B, lanes 1 and 2, we separately analyzed
normal and tumor TRs by two-dimensional gel electrophoresis. Comparison
of 75Se and protein profiles on two-dimensional gels for
normal and tumor samples suggested that all signals shown in Fig. 2
corresponded to TR1. TR3 was not detected on two-dimensional gels
because it was present in lower levels than TR1 and exhibited a
different isoelectric point.
The two-dimensional gel electrophoresis analysis (Fig. 2) revealed
dramatic heterogeneity within TR1 preparations from normal and tumor
samples. TR1 bands were separated on two-dimensional gels according to
both charge and mass. Nine representative bands from two-dimensional
gels for normal and tumor samples (Fig. 2, E and
F) were digested with trypsin. Tryptic digests were analyzed by MALDI time-of-flight mass-spectrometry (MALDI-TOF MS).
Experimentally determined peptide masses matched to the peptide masses
predicted from the tryptic digestion of a deduced mouse TR1 sequence
(see details of mouse TR1 sequences below) and covered >50% of the protein sequence (cysteine-containing and N-terminal peptides were not
detected). No post-translational modifications within TR1 that could
potentially contribute to different mobilities of TR1 forms on
two-dimensional gels were detected. In addition, no significant
differences in tryptic peptide maps were found between normal and tumor samples.
In addition to multiple TR1 forms that were resolved by two-dimensional
gel electrophoresis (Fig. 2) but migrated as a single 57-kDa band on
SDS-PAGE gels (Fig. 1B), we detected TR1 forms that
significantly differed from the 57-kDa isoforms. We noted that longer
exposure of 75Se signals detected with a PhosphorImager and
analysis of anti-TR1 immunoblot signals often produced two additional
bands, ~67 and ~110 kDa. These bands partially co-purified with the
57-kDa forms and were present as minor forms in apparently homogeneous
enzyme preparations. The 67- and 110-kDa species did not cross-react with anti-TR2 and anti-TR3 antibodies. Migration properties of the
110-kDa minor band corresponded to that of the TR1 homodimer, although
the biochemical basis for the possible dimer formation under these
conditions is not clear. The possible origin of the 67-kDa band is
discussed below.
Heterogeneity within Human Thioredoxin Reductase
Preparations--
We further tested if the extensive heterogeneity
observed within mouse TR1 preparations, occurs in human TR1
preparations. For this purpose, we selected a human T-cell line, JPX9,
that was previously used as a source of TR1 (12). To purify human TR1,
we initially utilized a previously developed three-step procedure (12).
The purified protein was analyzed by electrospray ionization (ESI) mass
spectrometry. The recorded ESI-MS spectra showed a broad peak centered
at 54,860 Da, that was 230 Da more than the mass predicted from the
previously reported human TR1 sequence (24). Reduction and
carboxymethylation of the enzyme increased its mass to 55,626 Da, but
did not significantly narrow the mass peak. The difference between
native and carboxymethylated forms, 762 Da, corresponded to 13.4 alkylated groups. The predicted human TR1 sequence contained 13 cysteines and 1 selenocysteine. Considering that isolated TR1 is often
contaminated with a truncated form lacking the last two residues,
Sec498 and Gly499, the data suggested
that the isolated TR1 could be fully alkylated with iodoacetic acid and
that cysteines and selenocysteine were not modified in the native enzyme.
We further took advantage of a recent observation that human TR1 could
be additionally fractionated on a heparin column (16-18). Indeed, two
peaks for TR1 were eluted from the column, which were detected as
280-nm absorbing peaks (Fig. 3). The two
peaks could also be detected due the absorbance of a flavin at 450 nm
and due to the presence of 75Se in the fractions.
Integration of two major TR1 peaks indicated that the second-eluted
peak (peak II) had 3.8 times higher selenium content (calculated
on the basis of 75Se radioactivity) per mg of protein
compared with the first-eluted peak (peak I). The possible reduced
selenium content of TR1 in peak I is similar to the previous finding of
a HeLa cell TR1 form that had ~0.5 selenium per protein subunit (17).
The previously isolated mixture of human T-cell TR1 forms had ~0.74
selenium per protein subunit (12), suggesting that it could be a
mixture of TR1 molecules with variable selenium content. Lower levels of selenium may be due to a loss of selenium from TR1 due to hydrolysis in the presence of oxygen or reactive oxygen species, or due to the
presence of a truncated form of TR1 lacking C-terminal selenocysteine and glycine residues.
The fractions corresponding to Peak I shown in Fig. 3 were pooled, as
were fractions corresponding to Peak II, and the two samples were
further analyzed by SDS-PAGE, immunoblot assays, and isoelectric
focusing (Fig. 4). Major protein forms in
each pool migrated as a 55- and 57-kDa pair of protein bands with
approximately similar staining intensity (Fig. 4A, left
panel). These forms were also detected by immunoblot and
PhosphorImager assays (Fig. 4A, middle and right
panels). This observation was in agreement with a previous finding
of two closely migrating TR1 forms isolated from human JPX9 cells (12).
In addition, the pair of 55- and 57-kDa bands was resolved on gels
either run in a nonreducing buffer, or following reduction with
dithiothreitol and alkylation with iodoacetic acid, or following
denaturation in 6 M guanidine HCl, reduction with
dithiothreitol and subsequent carboxymethylation (data not shown).
The 55- and 57-kDa bands were transferred onto a polyvinylidene
difluoride membrane, digested with trypsin, and masses of tryptic
peptides were determined with a MALDI-time of flight MS. Both bands in
the doublet produced essentially identical peptide maps and matched
with a set of the predicted TR1 tryptic peptides. Even in this
instance, we could not detect any Cys-containing peptide, along with
the N-terminal and C-terminal peptide. In addition to the 55/57-kDa
pair of bands in peaks I and II, a protein form migrating as a 110-kDa
band was observed in both TR1 samples (Fig. 4A). Like the
mouse 110-kDa band, this form corresponded in size to a possible TR1 dimer.
Peaks I and II differed in that a minor ~67-kDa species were present
in peak I, but absent in peak II (Fig. 4A). This TR1 form
was detected as a Coomassie Blue-stained band and as a
75Se-labeled band. It was also reactive in immunoblot
assays with anti-TR1 antibodies. Thus, the minor 67-kDa form of TR1 was
detected in both mouse and human preparations. In addition, several
other minor forms of TR1 were present in peak I of human TR1. Bands with masses higher than 110 kDa could potentially be formed by a dimer
formation involving the 67-kDa form.
The protein mixtures present in peaks I and II were further analyzed by
tryptic digests followed by determination of peptide masses by MALDI
MS. By matching experimentally observed peptide masses with those
predicted from an in silico tryptic digest of the previously
reported human TR1 sequence, we were able to cover over 70% of the
human TR1 sequence. Again, no peptide masses were detected that matched
Cys-containing, N-terminal and C-terminal peptides. Spectra obtained
from peak I and peak II were virtually identical. The only differences
detected were an ion signal at 2110 Da present only in peak I and
signals at 1446 and 1430 Da present only in peak II. These peptides
could not be assigned in the human TR1 sequence.
Despite the close similarity observed by SDS-PAGE and mass spectrometry
analyses between the various TR1 forms present in peaks I and II (Fig.
4A), these proteins had different migration properties when
analyzed by isoelectric focusing (Fig. 4B). Each form was
represented by several protein bands (Fig. 4B). Overall, the
data indicated the presence of multiple forms of human TR1 that
differed in molecular masses and isoelectric points.
Alternative First Exon Splicing Results in the Formation of Three
Distinct mRNA Isoforms of Mouse and Rat TR1--
Having determined
no post-translational modifications, we tested whether observed
heterogeneity within human and mouse TR1 preparations could be
explained by genetic variations within TR1 sequences. In particular,
the occurrence of the 67-kDa form of TR1, which was ~10 kDa larger
than the protein deduced from known mammalian TR1 cDNAs, could not
be easily explained without invoking genetic mechanisms. During the
time of our analysis, no mouse TR1 sequences were available in
GenBankTM. Therefore, we obtained a cDNA sequence of
mouse TR1 by sequencing multiple EST clones. Surprisingly, the final
3626-nucleotide cDNA sequence encoded a protein of 613 residues
that had a predicted mass of 67.1 kDa. The deduced polypeptide
contained the C-terminal penultimate selenocysteine residue encoded by
a TGA codon and all sequence features found in other animal TR
sequences (N-terminal disulfide active center, NADPH- and FAD-binding
domains and a dimer-interface domain). The predicted protein differed
from the previously reported 499-amino acid sequences for mammalian
TR1s (24, 25) in that the mouse protein had 114 additional N-terminal residues. The new N-terminal sequence had no homology to coding regions
of other proteins when analyzed using BLAST programs against nonredundant (NR) and EST data bases, except that it exhibited >70%
identity with sequences deduced from a rat genomic clone that contained
a partial sequence of the TR1 gene (accession number AF189711).
A second mouse TR1 sequence, which encoded a 499-amino acid protein,
has recently appeared in GenBankTM (accession number
AB027565). Comparison of two mouse TR1 cDNA sequences revealed
identity in >3 kilobases that spanned most of the coding region and
the entire 3'-untranslated region. These sequences, however, had no
homology in 5'-end sequences (Fig. 5).
Subsequent BLAST analyses of mouse dbEST revealed a mouse EST sequence
(accession number AI956288) corresponding to a third TR1 form that had
an additional distinct version of the 5'-end sequence, but was
identical with the two full-size cDNA sequences in the downstream
region (Fig. 5). The use of three independent 5' variants deviating
from each other at the same place within the nucleotide sequence was
reminiscent of the alternative use of three exons; that is, different
5' sequences corresponded to alternative first exons, while common
sequences corresponded to common downstream exons.
This proposition was consistent with the BLAST analysis of rat NR and
EST data bases using sequences for three forms of mouse TR1 mRNA.
It revealed three potential forms of rat TR1 mRNA that corresponded
to the three mouse mRNA forms (Fig. 5). Moreover, the availability
of the partial sequence for the rat TR1 gene allowed us to predict
partial features of an exon-intron structure within this region (Fig.
6). The first exon within the rat genomic sequence corresponded to the 5' end of our mouse TR1 cDNA sequence, whereas the second exon that was separated from the first exon by an
~1.5-kilobase long intron, corresponded to the first common exon
within the three forms.
The three TR1 forms, TR1-I, TR1-II, and TRI-III (Fig. 6), were
designated according to order of exons within genomic sequences, which
was derived from the following observations. The first exon of the
TR1-III sequence was present in the rat genomic clone that also
contained the downstream sequences common for three TR1 forms, whereas
the first exons of the TR1-I and TR1-II sequences were not present in
the rat genomic clone sequence. Thus, 5' exons of TR1-I and TR1-II were
upstream of the 5' exon of TR1-III. We also found several EST sequences
(accession numbers AI526517, AI527732, AI226627, AI787452, AI314145,
and AI315024) that contained partial sequences for first exons of TR1-I
and TR1-II as well as downstream sequences that were common for the three forms (Fig. 6). Within these ESTs, 5' sequences of TR1-I preceded
5' sequences of TR1-II. Thus, within the genomic sequences, the 5' exon
of TR1-I should be first, the 5' exon of TR1-II, second, and the 5'
exon of TR1-III, third, followed by a fourth exon that had sequences
common for the three TR1 forms.
Interestingly, ESTs that contained 5' sequences for TR1-I and TR-II had
an additional internal stretch of 176 nucleotides, which was 100%
identical to internal sequences of exon 4 of a mouse apolipoprotein E
(ApoE) gene. It should be noted that known TR1 (chromosome 10)
and ApoE (chromosome 7) genes in mice are located on different
chromosomes. If ESTs containing TR1 and ApoE sequences were formed by
alternative splicing, then one possible explanation for their formation
is that mice may have an additional ApoE gene or a pseudogene located
within the TR1 gene. Alternatively, ESTs containing a 176-nucleotide
stretch could be formed by possible novel genetic mechanisms (such as
"reverse splicing"), which would allow insertion of a DNA (or
mRNA) sequence within another DNA (or mRNA) sequence.
Alternative Use of First Exons within TR3 Genes--
The
observation of alternative mRNA forms of TR1 in rats and mice
prompted us to test whether this mechanism is used in genes for other
TRs. Indeed, computer search analysis of NR and EST data bases revealed
three alternative human TR3 transcripts (TR3-I, TR3-II, and TR3-III)
that differed in their 5' sequences (Fig. 7) and were formed by alternative first
exon splicing (Fig. 8). TR3-I (accession
numbers AF171054, AF044212, AF106697, and AB019694) was a previously
reported nucleus-encoded mitochondrial form of TR3 (5-10). The TR3-II
transcript (accession number AF166127) was a previously identified
isoform of human TR3 mRNA, SelZf2 (26). This transcript
appeared to be inhibitory for TR3 expression since it contained an
initiator ATG codon, followed by an in-frame stop signal, in a
different frame with established TR3 sequences. The TR3-III transcript
(accession number AB019695) encoded a protein that lacked a
mitochondrial signal but instead contained a 5-amino acid extension.
The lack of signal peptides in TR3-III suggested that the protein could
reside in the cytosol.
The human TR3 gene, located on chromosome 22 (accession number
AC000090), was previously sequenced by the Human Genome Project as
evidenced by the BLAST analysis of dbNR. Organization of the human TR3
gene and its upstream region are shown in Fig. 8. Comparison of
cDNA and genomic sequences indicated that alternative TR3-I,
TR3-II, and TR3-III transcripts were formed due to alternative use of
three 5' exons within the gene. We also analyzed partial sequences of
the mouse TR3 gene (accession number AC003066, clone tbx1, strain
129X1/SvJ ES, from cell line RW4, located on chromosome 16) that were
generated by the Mouse Genome Project and partial rat TR3 genomic
sequences that were found in the genomic clone containing an upstream
region of the COMT gene. Analyses of these sequences with an exon
finding program revealed the presence of a predicted conserved exon
that could generate TR3-III forms of mouse and rat TR3 (Fig. 7). Thus,
the use of alternative first exon splicing to form multiple TR3 forms
appeared to be conserved in humans, mice, and rats. However, no ESTs
that corresponded to mouse and rat TR3-III were detected.
We have previously noted that the human TR3 gene partially overlapped
with the gene for COMT (10). In the present study, we performed
detailed computer analysis of human TR3 and COMT genes and analyzed
whether this structural organization of these genes is conserved in
other animals. We found that the first exon of the human TR3 gene
overlapped with the first exon of the gene for membrane-bound (MB)-COMT
(Fig. 8). The mTR3 and MB-COMT genes were located on different DNA
strands on a chromosome 22q11.2 band. Translation of MB-COMT mRNA
resulted in the 30-kDa protein containing an N-terminal transmembrane
domain (27). Interestingly, the COMT gene, like the human TR3 gene, had
an additional transcription initiation site. The use of this site
resulted in a transcript that lacked the first two exons and translated
into a shorter 26-kDa soluble catechol-O-methyltransferase
(S-COMT) (27). Human DNA sequences containing exon 1 of the TR3-I gene
and exon 1 of the MB-COMT gene were well separated from other exons
within these genes. For example, transcription start sites for S-COMT
and TR3-III were separated by ~30 kilobases (Fig. 8).
Further analysis of mouse and rat TR3 genes revealed that exons
encoding a mitochondrial signal overlapped with the first exons of
mouse and rat MB-COMT genes. Conservation of structural organization of
TR3 and COMT genes in humans, mice, and rats suggested that certain TR3
and COMT forms could potentially exhibit parallel or contrasting
regulation of expression.
Alternative Use of First Exons in the Drosophila TR Gene--
In
addition to the finding of alternative first exons within mammalian TR
genes, we detected a Drosophila gene for TR that produced
two alternative transcripts (Fig. 9).
Like TR3 forms, these transcripts encoded proteins that differed in
their N-terminal regions. The previously reported Drosophila
TR sequence (accession number U81995) corresponded to a putative
cytosolic form of this enzyme, while the additional form of the protein
was evident from the BLAST analysis of dbEST and was represented by
several ESTs (e.g. accession numbers AA803764 and AA820592
and 6 other ESTs). This second form of the Drosophila TR
contained a predicted mitochondrial signal peptide. Thus, it is likely
that a single Drosophila TR gene encodes two proteins,
mitochondrial and cytosolic. Interestingly, although the order of
predicted mitochondrial and cytosolic exons was different, the location of the splicing site that separated variable and common sequences was
conserved between mammalian TR3 and Drosophila TR genes
(Fig. 9B).
We report in the present study that extensive heterogeneity exists
among animal TRs and that splicing of first exons of TR genes occurs as
a general genetic mechanism for producing multiple mRNA isoforms.
The data suggest that this evolutionary conserved genetic mechanism
contributes to heterogeneity that is observed among animal TRs.
The fact that human TR1 isolated from HeLa cells occurs in at least two
distinct forms has been established previously (16-18). The two forms
could be separated by affinity chromatography on a heparin column. The
first form was not retained on a heparin column, exhibited full
catalytic activity and selenium content and showed reactivity with
anti-rat liver TR1 antibodies. This form could be converted into the
high-affinity TR1 form upon reduction. An additional TR1 form was also
isolated that could bind the heparin column without prior reduction.
This form contained ~0.5 selenium per subunit and had reduced
catalytic activities and reactivity with anti-rat liver TR1 antibodies.
Our present study indicates that the number of TR1 forms is much larger
than previously thought and that these forms differ in electrophoretic
mobility on SDS-PAGE and isoelectrofocusing gels. In particular, minor
67-kDa isoforms of TR1 were detected in our study that migrated
significantly slower on SDS-PAGE gels than would be predicted from
previously reported gene sequences. To characterize various mouse TR1
forms, we used affinity chromatography on ADP-Sepharose. Interestingly,
this simple technique was sufficient to enrich TR1 to an extent that it
could be directly analyzed on two-dimensional gels and visualized by
protein staining. Since multiple forms of TR1 were visualized after
only a single isolation step, this procedure may provide a basis for
future high-throughput protein microchemistry analyses of TRs isolated
from multiple sources or under various conditions.
To define the basis for heterogeneity within human and mouse enzyme
preparations, we initially used protein microchemistry techniques and
established that detected tryptic peptides did not differ between
various enzyme isoforms that had different electrophoretic mobility on
polyacrylamide gels or were isolated from normal or malignant cells. It
remains to be determined what post-translational modifications, if any,
are involved in the formation of 55-57-kDa TR1 forms in humans and mice.
To further characterize a mechanism responsible for the formation of
multiple TR1 forms and, in particular, for the occurrence of 67-kDa TR1
species, we turned our attention to possible genetic mechanisms. Since
no mouse TR1 sequences were available in sequence data bases, we
initially determined the mouse TR1 cDNA sequence. Interestingly,
this cDNA encoded a protein of 67 kDa that was formed from a coding
region starting with an ATG codon (in a favorable Kozak consensus
sequence for phasing the message for translation) upstream of the
previously predicted translation start site. The predicted extended
N-terminal domain had no homology to known protein sequences, and at
present its function is not known.
Recently, several new mammalian TR1 cDNA sequences, including one
for mouse TR1 (28), were deposited into GenBankTM.
Examination of NR and EST sequences and their comparison to the mouse
TR1 cDNA sequence that was determined in the present study,
revealed three forms of mouse and rat TR1 cDNAs that had unrelated
5' sequences, but common downstream sequences. We also observed three
human TR1 forms (BE618239, AU077310, and previously known TR1
sequences) obtained by alternative exon splicing. The location of an
alternative splicing site was conserved among mouse, rat, and human TR1 genes.
Alternative first exon splicing that was found to be used to synthesize
alternative mRNA forms of mouse and rat TR1 mRNA is a
previously recognized genetic phenomenon (29). It is used to
accommodate requirements for elevated expression of a protein in a
tissue-specific manner and to synthesize protein species that differ in
their intracellular location (e.g. alternative forms may
contain or lack an organelle-targeting signal peptide or a
trans-membrane domain). In addition, alternative first exon splicing
may be used as an on/off switch, which functions by alternative expression of protein-expressing and protein-suppressing mRNA forms. In this case, the "off" form would contain additional
upstream initiation sites followed by stop signals that would suppress protein expression, whereas the "on" form would produce a
functional protein.
Well characterized examples of alternative first exon splicing include
genes of enzymes that are involved in "liver-associated" xenobiotic
metabolism and detoxification. The human microsomal glutathione
S-transferase utilizes four alternative first exons, two of
which appear to be functional and result in mRNA sequences differing in the 5'-untranslated region, and two nonfunctional forms as
they contain additional upstream out of frame initiation signals (30).
These four mRNA forms were predicted to be generated from a common
promoter. The rat One interesting observation made in the present study is that several
mouse ESTs contained apolipoprotein E sequences within the TR1
sequences. Since all these ESTs originate from the same cDNA
library (Sugano mouse kidney mkia), additional sequences from other
sources may be required to confirm the observed puzzling location of
the ApoE sequences. If confirmed, the finding that an ApoE gene is
located within the mouse TR1 gene may have important biomedical
implications. Previous molecular genetic studies identified three genes
(presenilin 1, presenilin 2, and Having determined the use of alternative first exon splicing in
regulation of expression of mouse and rat TR1 mRNAs, we tested whether this mechanism is used to express multiple forms of other TRs.
Indeed, two unrelated 5'-end sequences, followed by common downstream
sequences were evident from the analysis of two human TR2 cDNA
sequences (EST sequence, accession number AA460989, and a cDNA
sequence, accession number AF133519). However, the lack of full-size
cDNAs and genomic sequences for mammalian TR2 did not allow us to
evaluate the significance of this finding.
The analysis of mammalian TR3 genes was more fruitful and revealed a
remarkable genetic complexity within genomic segments that contained
TR3 genes. Like mouse and rat TR1 genes, the human TR3 gene contained
three alternative first exons. However, the previously defined
translation initiation site was located upstream of the first common
exon and thus the use of alternative first exons resulted in different
N-terminal sequences of the protein. The use of exon 1 resulted in the
formation of a mitochondrial form of the enzyme, TR1-I (exon 1 encoded
a mitochondrial signal peptide). The use of exon 2 instead of exon 1 generated an mRNA form (TR3-II) that contained an upstream out of
frame initiation site followed by a termination signal. This form could
potentially be used as an off switch. If exon 3 was used in
place of either exon 1 or exon 2, the N-terminal sequence of TR3 was
extended by only 5 residues. The lack of signal sequences within
TR3-III suggested that this protein could be located in the cytosol.
Such a complex organization of TR3 genes was even more complicated by
the fact that exon 1 of human, mouse, and rat TR3 genes overlapped with
exon 1 of the MB-COMT gene located on a complementary DNA strand. It
has previously been established that the COMT gene gives rise to two
alternative protein forms, a membrane-bound and a soluble form (27).
Only the MB-COMT mRNA had common sequences with TR3 mRNA, and
only TR3-I mRNA (mitochondrial form) overlapped with MB-COMT
mRNA. Interestingly, the COMT knockout mice have been previously
generated and found to exhibit sexually dimorphic changes in
catecholamine levels and behavior (35). No sufficient experimental
details were reported (35) to evaluate whether knockouts could have
affected expression of some of the TR3 forms. It is also not known
whether TR3 could be involved in psychiatric disorders and symptoms,
including the psychopathology associated with the 22q11 microdeletion,
the area of the human chromosome 22, where the COMT and TR3 genes are located.
Availability of the completely sequenced Drosophila genome
and a large number of EST sequences allowed us to test if alternative first exon splicing is also used in Drosophila. We
identified two alternative exons within the Drosophila TR
gene that generated predicted mitochondrial and cytosolic enzymes.
Interestingly, the site for junction of alternative first exons and a
common downstream exon was conserved between mammalian TR3 and
Drosophila TR genes, suggesting a close evolutionary
relation between these genes. In addition, homology and phylogenetic
analyses (data not shown) indicated slightly higher evolutionary
linkage between Drosophila TR and mammalian TR3 genes than
between mammalian TR1 and TR3 genes.
In conclusion, we identified alternative exon splicing as a general
mechanism to express multiple mRNA forms of animal TRs. Such
mRNA forms encoded proteins that have common or different N-terminal sequences and that were responsible, at least in part, for
observed heterogeneity within animal TR preparations. The use of
alternative first exon splicing has not been described previously for
any known selenoprotein. Further studies are required to determine the
functional role of alternative first exon splicing in regulation of
expression and function of animal thioredoxin reductases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(5) and TrxR2 (6)), like TR1,
appeared to be ubiquitously expressed (5-10). This enzyme was
described as a mitochondrial thioredoxin reductase because it was shown
to contain a mitochondrial signal peptide. In addition, this protein
was localized in mitochondria by detecting various transiently
expressed, tagged forms of TR3 and by immunoblot assays with antibodies
specific for TR3 (5-10). TR2 had not been extensively characterized
nor its full amino acid sequence reported (10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF
)/c-myc double transgenic mice
(6-15 months old) were maintained and labeled with 75Se,
and their livers and liver tumors were dissected as described previously (19). The coexpression of TGF
and c-Myc results in
multiple liver tumor formation by 6 months of age. Three unlabeled wild
type mouse livers (4 g) and a single 75Se-labeled wild type
liver (0.5 g) were mixed and homogenized in 10 ml of 40 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.6 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 µg/ml
aprotinin, and 5 µg/ml leupeptin (buffer A). The homogenate was
centrifuged at 18,000 rpm for 20 min, and the supernatant was loaded
directly onto a 3-ml ADP-Sepharose (Amersham Pharmacia Biotech) column. The column was washed extensively with 150 mM NaCl and 40 mM Tris-HCl, pH 7.5, and proteins were eluted with 1 M NaCl in 40 mM Tris-HCl, pH 7.5. Protein
fractions were tested by SDS-PAGE and immunoblot analyses with
antibodies specific for TR1 and TR3 (10), pooled, and further analyzed
by a two-dimensional gel electrophoresis. TR1 was also isolated from
TGF
/c-myc tumors using the same procedure except that 5 unlabeled liver tumors (11.5 g) and one 75Se-labeled liver
tumor (3.5 g) were homogenized in 15 ml of buffer A.
-counter. JPX9
cells were grown on an RPMI 1640 medium in the presence of 10% fetal
bovine serum and metabolically labeled with 75Se as
described previously (12). Human A431 cell line was grown as described
(10) and TRs were purified from sonicated crude extracts on an
ADP-Sepharose column as described for mouse enzymes.
-cyano-4-hydroxy cinnamic acid
matrix (10 mg/ml in acetonitrile/trifluoroacetic acid, 0.1%) and
allowed to air-dry before data collection. Spectra were acquired on a Voyager RP mass spectrometer using oxidized bovine insulin
chain as
internal standard for calibration (21, 22).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/c-myc double transgenic mice. To minimize potential
losses of certain TR forms due to different elution profiles of these
forms on columns commonly used in TR isolation procedures, we utilized
only a single step, an affinity ADP-Sepharose chromatography, to purify TRs.
/c-myc
mice were compared with liver tumors from the same mice (19). On the
other hand, the latter studies found that expression of TR1 was
slightly increased in liver tumors relative to surrounding normal
livers in TGF
/c-myc mice (19). Fig. 1B shows
immunoblot analyses of mouse TR1 and TR3 enriched from normal wild type
and transgenic malignant livers on an ADP-Sepharose column. Proteins
isolated from these two sources migrated similarly on SDS-PAGE. TR1 had
a molecular mass of ~57 kDa, whereas TR3 migrated as an ~55-kDa
protein. Although TR1 is more abundant than TR3 in liver (16), the
ratio of these enzymes was approximately the same in wild type and
tumor samples (Fig. 2B),
suggesting that expression of TR3, like that of TR1 (but in contrast to
GPx1), was unchanged or perhaps somewhat elevated during malignant
transformation in TGF
/c-myc mice.
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Fig. 1.
Isolation and characterization of mouse liver
thioredoxin reductases. A, detection of
75Se by PhosphorImager analysis in fractions from an
ADP-Sepharose column. 75Se-Labeled homogenates from wild
type livers (lanes 1-3) and hepatocarcinomas of
TGF /c-myc transgenic mice (lanes 4-6) were
fractionated on an ADP-Sepharose column. Flow-through (lanes
1 and 4), low-salt wash (lanes 2 and
5), and high-salt elution (fractions 3 and 6) fractions were
analyzed by SDS-PAGE analysis. The positions of TR (mixture of TR1 and
TR3), GPx1 and GPx4 are indicated on the right. B,
immunoblot analyses of mammalian thioredoxin reductases. Wild type
liver TR fractions (A, lane 3) and liver tumor TR
fractions (A, lane 6) were probed with antibodies specific
for TR1 (upper panel). The blot was then stripped and
reprobed with antibodies specific for TR3 (lower panel).
Also analyzed were a homogenous preparation of a rat prostate TR1
obtained by a three-step procedure, employing DEAE-Sepharose,
ADP-Sepharose, and phenyl-HPLC columns (shown as Rat prostate,
lane 3) and a human TR preparation obtained by affinity
chromatography of a homogenate from A431 cells on an ADP-Sepharose
column (shown as Human A431 cells, lane 4). The rat sample
did not contain TR3, which had different elution profiles than TR1 from
DEAE-Sepharose and phenyl-HPLC columns. The positions of the 57-kDa
form of TR1 and the 55-kDa form of TR3 are indicated on the
right.
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Fig. 2.
Two-dimensional gel electrophoresis of mouse
TR preparations. Wild type liver and TGF /c-myc liver
tumor TR preparations were subjected to two-dimensional gel
electrophoresis analysis, followed by detection of 75Se by
PhosphorImager analysis (A-D) and staining of proteins with
Ponceau S (E and F). Panels A and
B show full two-dimensional gels, whereas panels
C-F show enlarged areas that correspond to 75Se
signals on panels A and B. Panels A, C, and
E show TR1 isolated from wild type livers, and panels
B, D, and F, from liver tumors.
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Fig. 3.
Fractionation of human TR1 preparations on a
heparin column. TR1 was first isolated from
75Se-labeled human JPX9 cells using a three-step procedure
(12). The enzyme was then further fractionated on a heparin-HPLC
column. Proteins eluted were detected by absorbance at 450 (due to the
presence of flavin and shown as the smaller signal on the upper
panel) and 280 nm (shown as the larger signal on the upper
panel) and by detection of 75Se in fractions from the
column using a -counter (lower panel). For subsequent
analyses, fractions corresponding to peak I were pooled, as were those
corresponding to peak II, and further analyzed.
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Fig. 4.
Characterization of human TR1 preparations by
gel electrophoresis. A, SDS-PAGE analyses. Two TR1
preparations (peaks I and II; see Fig. 3) were subjected to
SDS-PAGE analyses followed by detection of proteins by Coomassie Blue
staining (left panel), immunoblot analyses with antibodies
specific for TR1 (middle panel), and detection of
75Se by PhosphorImager analyses (right panel).
For better visualization of protein, immunoblot and 75Se
signals, three times more material was loaded on the gel from peak I
than from peak II. The positions of the 55- and 110-kDa forms are
indicated on the left. B, isoelectric focusing. Two TR1
preparations (peaks I and II) were subjected to
isoelectric focusing followed by detection of proteins by Coomassie
Blue staining (left panel) or detection of 75Se
by PhosphorImager analyses (right panel). The positions
and apparent isoelectric points of three standards are shown on the
left.
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Fig. 5.
Nucleotide sequence comparisons of TR1 forms
obtained by alternative first exon splicing in mice and rats. Each
of the three forms was represented by mouse and rat sequences. The
three forms had distinct 5' sequences (exons 1, 2, or
3), but shared downstream sequences (exon 4).
Shown in this figure are: MTR1-I, first form of mouse TR1 (accession
number AB027565, dbNR cDNA sequence); MTR1-II, second form of mouse
TR1 (accession number AI956288, dbEST sequence); MTR1-III, third form
of mouse TR1 (this work); RTR1-I, first form of rat TR1 (accession
number AF220761, dbNR cDNA sequence); RTR1-II, second form of rat
TR1 (accession number AF108213, dbNR cDNA sequence); RTR1-III,
third form of rat TR1 (accession number AF189711, dbNR genomic
sequence); and RTR1 gene, partial sequence of the rat TR1 gene
(accession number AF189711, dbNR genomic sequence). ATG codons that
correspond to the previously predicted initiation site within mammalian
TR1 sequences are shown in uppercase. Intronic sequence is
boxed. The vertical line indicates sites for junction of
exons 1, 2, or 3 to exon 4. Numbers on the left
correspond to nucleotide numbers for GenBankTM sequences
shown in the figure.
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Fig. 6.
Predicted structural organization of mouse
and rat TR1 genes. Upper portion of the figure shows organization
of the TR1 gene in mice and rats. Three alternative forms of TR1
mRNA are obtained from a single TR1 gene by initiating
transcription at exons 1, 2, or 3 (indicated by numbers
above exon boxes), followed by alternative splicing to link a used exon
to exon 4. The downstream organization of the gene is not known (shown
as question mark). ATG codons indicate alternative
translation initiation sites. Filled boxes indicate
translated sequences. Open boxes correspond to untranslated
regions. Lower portion of the figure shows organization of several ESTs
(accession numbers AI526517, AI527732, AI226627, AI787452, AI314145,
and AI315024) that were used to determine the order of exons in the TR1
gene. These ESTs contained sequences that were identical with partial
sequences of exon 1, exon 2, and the last exon of the TR1 gene. In
addition, ESTs contained an internal stretch of sequences identical
with internal sequences of exon 4 of apolipoprotein E gene (shown as
ApoE in the upper portion of the figure).
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Fig. 7.
Sequence comparisons of TR3 forms obtained by
alternative first exon splicing in mice, rats, and humans.
A, nucleotide sequence comparisons of three alternative TR3
cDNA forms. The three forms utilize alternative first exons
(exons 1, 2, or 3), followed by the common exon
(exon 4). Exon-intron splicing sites are shown by
interruptions in horizontal lines. Exonic sequences are
shown in uppercase, and intronic sequences, in
lowercase. Numbers on the left and
right indicate nucleotide numbers within
GenBankTM genomic sequences that were used to assemble the
figure. Numbers within sequences indicate numbers of
intronic nucleotides present within sequences shown in the figure. ATG
codons that are predicted to initiate translation of TR3-III mRNAs
are shown in bold. These are characterized by favorable
Kozak consensus sequences for phasing mRNA for initiation of
translation. The occurrence of exons 2 and 3 in human sequences is
indicated by the analysis of dbNR. The occurrence of exon 3 in mouse
and rat sequences is predicted in this study based on the analysis of
mouse and rat genomic sequences with an exon-searching program.
Sequences shown are from the following dbNR genomic sequences: HTR3-I,
first form of human TR3 (accession number AC000090), MTR3-I, first form
of mouse TR3 (accession number AC003066), RTR3-I, first form of rat TR3
(accession number RN5CATOMT), HTR3-II, second form of human TR3
(accession number AC000090), HTR3-III, third form of human TR3
(accession number AC000090); MTR3-III, third form of mouse TR3
(accession number AC003066); RTR3-III, third form of rat TR3 (accession
number RN5CATOMT). B, amino acid sequence comparisons of
TR3-I and TR3-III. Same sequences were used here as in Fig.
7A. Exon-intron junctions (indicated by interruptions within
horizontal lines) were predicted with an exon finding
program and by matching cDNA and genomic sequences.
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Fig. 8.
Structural organization of human TR3 and COMT
genes. TR3 and COMT genes are located on complementary DNA stands
and overlap in their first exons such that transcription within the
membrane-bound form (MB-COMT) of the COMT gene is initiated within the
coding region of the mitochondrial (TR3-I) form of the TR3 gene.
However, transcription start sites in the TR3-III gene and a soluble
form (S-COMT) of the COMT gene are separated by ~30
kilobases. Alternative first exon splicing is used in the TR3 gene.
TR3-I mRNA is composed of 6 exons (exons 1 and 4-8); TR3-II
mRNA, 6 exons (exons 2 and 4-8); TR3-III mRNA, 6 exons (3-8);
MB-COMT mRNA, 6 exons (exons 1-6); and S-COMT mRNA, 4 exons
(exons 3-6). Translation start sites within TR3 and COMT genes are
indicated by ATG codons. Initiator ATG codon for TR3-I mRNA is
located within exon 1, and for TR3-III mRNA, within exon 2. Translation start site for TR3-II is located within exon 2, but
followed by an in-frame stop signal (not shown). The translation
initiation site in the MB-COMT mRNA is the first ATG within exon 3, and in the S-COMT mRNA, the second ATG within exon 3. Filled
boxes indicate translated sequences. Open boxes
correspond to untranslated regions.
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Fig. 9.
Alternative forms of Drosophila
TR. A, structural organization of the
Drosophila TR gene. Each numbered box represents
an exon within the gene. Open boxes show untranslated
regions, and filled boxes, coding regions. There are two
alternative forms of Drosophila TR mRNA that are encoded
by either exons 1 and 3-5 (DTR-I, accession number U81995), or exons
2-5 (DTR-II; accession number AA803764 and AA820592). B,
amino acid sequences of N-terminal regions of two alternative
Drosophila TR sequences. Human TR3-I and TR3-III, which
deviate from each other at the same site within the intron-exon
structure, are shown for comparison. Boxed residues
represent predicted mitochondrial signal peptides. The vertical
line represents exon-intron junctions. Identical residues within
four sequences are highlighted. The horizontal
line over the sequences shows the location of the disulfide active
center.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamyltransferase gene also has four
alternative first exons but utilizes four alternative promoters to form
mRNA species differing in the 5'-untranslated region but encoding
identical proteins (31). Additional examples of alternative first exon
splicing include family 19 of the cytochrome P450 gene superfamily (32)
and the UDP-glucoronosyltransferase gene (33), which contain numerous
first exons but a common splice junction located upstream of the
translation start site.
-amyloid precursor protein)
associated with early onset of Alzheimer's disease (AD), and one gene
(ApoE) associated with late onset of AD (34). An additional AD
susceptibility locus has been mapped to a broad region of chromosome
12, but the gene responsible for a defect has not been identified.
Whereas the human ApoE gene resides on chromosome 19, the TR1 gene is
located on human chromosome 12. If a mouse TR1 gene indeed contains an
additional ApoE gene and this gene organization is conserved in humans,
then a new ApoE gene may provide an additional susceptibility marker
for AD. Alternatively, the ApoE sequences located within the mouse TR1
gene may be a part of an ApoE pseudogene, or the internal ApoE
sequences could have been incorporated into TR1 sequences by an unknown
genetic mechanism. To confirm the predicted organization of mouse and rat TR1 genes and to determine the actual mechanism involved in linking
TR1 and ApoE sequences, we have to wait until actual sequences of TR1
genes are available.
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Addendum |
---|
A recent paper by Rundlof et al. (36) also reported identification of three variants of rodent TR1 mRNA.
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FOOTNOTES |
---|
* 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 mouse TR1 cDNA sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number AF333036.
** To whom correspondence should be addressed. Fax: 402-472-7842; E-mail: vgladyshev1@unl.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M004750200
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ABBREVIATIONS |
---|
The abbreviations used are:
TR, thioredoxin
reductase;
TGF, transforming growth factor
;
PAGE, polyacrylamide
gel electrophoresis;
HPLC, high performance liquid chromatography;
MALDI, matrix-assisted laser desorption ionization;
GPx, glutathione
peroxidase;
Sec, selenocysteine;
NR, nonredundant;
COMT, catechol-O-methyltransferase;
MB-COMT, membrane-bound
catechol-O-methyltransferase;
S-COMT, soluble
catechol-O-methyltransferase;
AD, Alzheimer's
disease.
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