Download cDNA cloning, expression and chromosomal localization of the

Genomic structure and chromosomal localization of
human thioredoxin-like protein gene (txl).
Antonio Miranda-Vizuete and Giannis Spyrou#
Department of Biosciences at Novum, Karolinska Institute, S-141 57
Huddinge, Sweden
# To whom correspondence should be addressed: Dept. of Biosciences, Center for
Biotechnology, Karolinska Institutet, Novum, S-141 57 Huddinge, Sweden.
Tel.: 46-8-6089162; Fax: 46-8-7745538; Email: [email protected]
Abbreviations: EST, expressed sequence tag; STS, sequenced tagged site; Trx,
thioredoxin; UTR, untranslated region.
1
The sequence data reported in this paper have been submitted to the GenBank Data
Libraries under the accession numbers: AF143890 to AF143897 for human txl exons;
AF143404 and AF143405 for the putative txl from D. melanogaster and C. elegans,
respectively; and AF143898 to AF143900 for C. elegans txl exons.
2
SUMMARY
Human thioredoxin-like protein (txl) is a novel member of the expanding
thioredoxin superfamily whose main characteristic is the presence, after the
thioredoxin domain, of a C-terminal extension of 184 residues with no homology
with any other protein in the databases. Txl is a cytosolic ubiquitously expressed
protein and it has been copurified with a kinase of the STE20 family, which is
proteolytically activated by caspases in apoptosis. However, no cellular function
has yet been assigned to this protein. In the present study we report the genomic
organization of the txl gene which encompasses approximately 36 kb organized in
eight exons ranging from 96 bp to 303 bp. In contrast, intron sizes are much bigger
ranging from 1.5 kb to 12 kb. Chromosomal localization of txl gene revealed that it
maps at position 18q21, a region frequently affected in different human tumours.
Furthermore, we have identified the putative homologues of txl in both Drosophila
melanogaster and Caenorhabditis elegans that display much closer homology to the
known thioredoxins than the human txl protein. Indeed, critical residues for
optimal thioredoxin activity are present in both Drosophila and Caenorhabditis txl
but absent in the human protein suggesting that txl might have evolved to carry
out a function different from the general disulfide reductase typical of
thioredoxins.
3
Thioredoxins (Trx) are small, ubiquitous proteins that act as general proteindisulfide reductases via reversible oxidation of their conserved active site Trp-CysGly-Pro-Cys (WCGPC)). Oxidized thioredoxin is reduced by the flavoenzyme
thioredoxin reductase to its active form (Holmgren, 1985). Thioredoxin is a very
compact protein with a globular three-dimensional structure constituted by a
central core of five strands of -sheet enclosed by four -helices with the active site
located on a protrusion of the protein (Eklund et al., 1991). So far, only two
thioredoxins have been described in mammalian cells: Trx1 is a cytosolic protein
with many different functions most of them depending on the redox active site
(Holmgren, 1985) and Trx2 is a mitocondrial protein whose only function so far
described is to act as a electron donor for a mitochondrial thioredoxin-dependent
peroxidase, thus being a major antioxidant defense in this organella (Spyrou et al.,
1997; Araki et al., 1999). In addition, some other proteins contain thioredoxin
modules or domains in which some residues within the active site are changed. For
example, PDI (protein disulfide isomerase) is an ER-resident protein constituted by
four independent thioredoxin domains, two of them which have an active site
where a proline residue is changed to a hystidine (WCGHC) and the other two
domains with no active site but still maintaining the thioredoxin folding (Kemmink
et al., 1997). Other ER-resident proteins like calcium binding protein-1 (CaBP1) and
phospholipase C- (PLC-contain two thioredoxin domains with the same
substitution at the active site as PDI (Fullekrug et al., 1994; Bennett et al., 1988). The
thioredoxin domain is also present in a nuclear protein termed nucleoredoxin in
4
which the glycine residue of the active site is changed to proline (WCPPC)
(Kurooka et al., 1997).
We recently described a novel member of the thioredoxin superfamily that we
named txl (thioredoxin-like protein) that contains an extension of 184 residues at
the C-terminus of the thioredoxin domain with no homology with any other
protein in the databases. The protein is not a substrate for thioredoxin reductase,
the physiological electron donor of all thioredoxins, and its mRNA is expressed in
all the tissues investigated (Miranda-Vizuete et al., 1998). Later, Lee et al. identified
the same protein (which they named TRP32) copurifying with a kinase that is
proteolytically activated by caspases in apoptosis. They showed that txl is able to
display some redox activity when DTT is used as electron donor with a truncated
form expressing only the thioredoxin domain (Lee et al., 1998). Thus, it seemed that
the C-terminal extension might regulate the activity of the thioredoxin domain
although the same truncated domain is not able to be reduced by thioredoxin
reductase (Miranda-Vizuete et al., 1998). As noted before, the main characteristic of
thioredoxins is the conserved sequence of their active site Trp-Cys-Gly-Pro-Cys.
Txl has a glycine residue instead of tryptophan and we speculated that this might
be the reason for the lack of activity with thioredoxin reductase as the tryptophan
residue has been described to stabilize the three-dimensional structure of the active
site (Eklund et al., 1991). Taken together, these data suggest that the cellular
function of txl might be not dependent of its redox properties.
In order to provide more insight into this novel member of the thioredoxin
superfamily, we have determined its genomic structure and assigned its gene
5
location at chromosome 18q21. We initiated our study identifying a partial
genomic sequence (accession number AC007052) that displayed homology when
compared with txl cDNA sequence. A detailed analysis of this entry showed that it
contained the whole txl cDNA sequence structured in eight exons and seven
introns spanning about 36 kb (Fig. 1). The position and size of the exons and
introns as well as the sequence of the intron/exon junctions and intron frames are
indicated in Table 1. The sizes of the exons are quite small, ranging from 96 bp to
303 bp. The first exon is the largest one and comprises of the 5´-UTR and the first 32
residues just before the active site. Exons 2 and 3 complete the thioredoxin domain
and exons 3 to 8 comprise the C-terminus of the protein. Exon 8 also contains the
3´-UTR. The genomic organization of the thioredoxin domain of txl is very similar
to the one of mitochondrial thioredoxin that is also organized in three exons1 while
it differs from the cytosolic thioredoxin as this one is organized in five exons
(Tonissen and Wells, 1991). In contrast to exon size, txl introns are much larger
ranging from 1.5 to 12 kb and all of them conform to the GT/AG rule for consensus
splice junction sequences (Breathnach and Chambon, 1981) . Introns 2,3,4,6 and 7
belong to type 0 where introns occur between codons, intron 5 is type 1
interrupting the first and the second base of the codon and intron 1 is type 2
interrupting the second and the third base of the codon. In summary, the structural
characterization of the gene encoding human thioredoxin-like protein reveals both
similarities and differences with the structure of other thioredoxin genes.
To assign the chromosomal localization of txl gene we searched the STS
(sequenced tagged sites) deposited at the GenBankTM against txl cDNA and
1
A.E. Damdimopoulos, manuscript in preparation.
6
identified one STS entry (STSG4286) that matched from base 1041 to 1165. This STS
has been mapped between the markers D18S474 and D18S64 by PCR screening of a
human-rodent radiation hybrid panel. These gene markers are located on
chromosome 18 at 71-83 centimorgans from the top of the linkage group. We
compared this location with the locations of other genes that have been mapped in
the region and show that the position of the human txl gene can be assigned to
chromosome 18q21 (Figure 2). This region of chromosome 18 has been extensively
studied because its involvement in different types of tumors (Uchida et al., 1996;
Ueda et al., 1997; Eppert et al., 1996). Bcl-2, an apoptosis inhibitor which was
originally discovered as a proto-oncogene that is activated in lymphomas by
translocation that results in its overexpression, also maps in the same region (Ohno
et al., 1987). Thioredoxin has been involved in tumor progression as it has been
found to be overexpressed in many human cancers and stimulates the proliferation
of a wide variety of human solid tumors and leukemic cancer cell lines.
Furthermore, a dominant-negative thioredoxin with its active site mutated is able
to revert the transformed phenotype of human breast cancer thus suggesting that
redox activity of thioredoxin is necessary for proliferation (Gallegos et al., 1996;
Baker et al., 1997). The mechanism by which thioredoxin promotes tumor
progression is not fully understood although stable cell lines expressing
thioredoxin are protected from apoptosis (Baker et al., 1997). In this context, there is
a primary line of evidence of how thioredoxin could be involved in this process as
it can inhibit apoptosis by direct association with ASK-1, an apoptosis signalregulating kinase, and dependent of the status of its redox active site (Saitoh et al.,
7
1998). The fact that human txl is copurified with a protein involved in apoptosis
(Lee et al., 1998) suggests that txl could also be involved in this process.
We used the human txl protein sequence to search for putative homologues in
Drosophila melanogaster and Caernorhabditis elegans. We identified two overlapping
EST entries in a Drosophila database (AI402225 and AI402230) that when
translated, resulted in a similar protein to that of human txl (accession number
AF143404). Similarly, the search in a Caenorhabditis database identified a genomic
clone (Y54E10.contig235) from chromosome I that displays high homology to
partial sequences of human txl protein. By computer assisted analysis of the
sequence we were able to identify the putative C. elegans
homologue of txl
(accession number AF143405) and its genomic organization in three exons of 267,
499 and 247 bp respectively, separated by two exons of 1912 and 871 bp and both
conforming the GT/AG rule. A comparison of the D. melanogaster and C. elegans txl
together with human txl and Trx1 is shown in Figure 3. From the comparison of all
these proteins, a very interesting pattern emerges as Drosophila and Caenorhabditis
txl thioredoxin domains are much closer to human Trx1 than human txl. Indeed,
critical conserved residues for optimal thioredoxin activity are absent in the human
txl protein while they are present in the others txl members (Figure 3). For instance,
Asp26 (all numbering referred to human Trx1) which participates in the charge
stabilization of the active site by its negative charge is changed to a Lys residue
with positive charge. Ala29 interacts by Van der Waals contact to Trp31 to stabilize
the active site surface (Eklund et al., 1991). In human txl these two residues are
changed to Met and Gly, respectively, and would not behave as the Ala-Trp pair.
8
Finally, the other residue flanking the active site, Lys36 that stabilizes the thiolate
in the active site is changed to a Leu residue with no charge and thus unable to
stabilize the thiolate intermediate (Eklund et al., 1991). These substitutions would
then explain the inability of human txl to be reduced by its physiological reductant
thioredoxin reductase (Miranda-Vizuete et al., 1998). It would be of great
importance the purification of any of the other txl homologues and to evaluate
their reducing activity in the presence of thioredoxin reductase. The fact that txl
seems to be present in lower eukaryotes but with important differences compared
to the human protein strongly suggest the possibility that human txl had evolved
to display a different function probably not dependent on its redox capacities. Its
implication in apoptosis should be considered as human txl copurifies with a
kinase of the STE20 family which is activated by caspases in apoptosis (Lee et al.,
1998). Indeed, yeast do not have txl homologue neither developed apoptosis
further supporting this possibility. Additional work is required to elucidate this
questions.
9
ACKNOWLEDGEMENTS
This work was supported by grants from the Swedish Medical Research
Council (Project 13X-10370) and the TMR Marie Curie Research Training Grants
(contract ERBFMBICT972824).
10
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LEGENDS TO THE FIGURES
Figure 1. Genomic organization scheme of human txl gene. The gene is
divided into eight exons and seven introns distributed over approximately 36 kb.
Exons are shown as solid bars while open bars represent the 5´-UTR and 3´-UTR
respectively.
Figure 2. Chromosomal localization of human txl gene. The human txl gene
is located at 71-83 centimorgans from the top of the linkage group of chromosome
18. By comparing this location with other genes in the region we have mapped it to
18q21.
Figure 3. Alignment of predicted amino acid sequence of human txl with
the putative D. melanogaster and C. elegans homologues and human Trx1.
Identical residues are indicated in black boxes. Asterisks indicate the critical
residues for thioredoxin activity that are not conserved in human txl. Human txl
sequence was used as reference for the identity/similarity (Id/Si) values.
15