I I N A S e y ~ w t w , 2000, Vol. 10(h), pp. 419-424 Reprint5 available directly from the publisher Photocopying permitted by license only

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Short Communication

Genornic Structure and Chromosomal Localization of Human Thioredoxin-Like Protein Gene (fxl) ANTONIO MIRANDA-VIZUETE and GIANNIS SPYROU*

D e p r t n r m t ofBzosciciices flt Noouin, Karolinska Institute, 5-143 57 Huddinge, Srilederr

(Reccioed \ f 4 h / 28, 19991

Human thioredoxin-like protein (txl) is a novel mem- ber of the expanding thioredoxin superfamily whose main characteristic is the presence, after the thiore- doxin domain, of a C-terminal extension of 184 resi- dues 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 organ- ized 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 fre- quently affected in different human tumours. Further- more, we have identified the putative homologues of txl in both Drosoplzila rnelanogastev and Caenorhabditis elegans that display much closer homology to the known thioredoxins than the human txl protein. Indeed, critical residues for optimal thioredoxin activ- ity 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 thiore- doxins.

Abhrer~intions: EST, expressed sequence tag, STS, sequenced tagged site, Trx, thioredoxin, UTR, untranslated region

Dntnbrise Accession No. AF143890 to AF143897 for human txl exons; AF143404 and AF143405 for the putative txl from D. melanogaster and C. ekgaiis, respectively and AF143898 to AF143900 for C. elegans txl exons:

Thioredoxins (Tm) are small, ubiquitous proteins that act as general protein-disulfide reductases via reversible oxidation of their conserved active site Trp-Cys-Gly-Pro-Cys (WCGPC). Oxidized thiore- doxin is reduced by the flavoenzyme thioredoxin reductase to its active form (Holmgren, 1985). Thoredoxin is a very compact protein with a globular three-dimensional structure constituted by a central core of five strands of P-sheet enclosed by four a-helices with the active site located on a protrusion of the protein (Eklund ef al., 1991). So far, only two thioredoxins have been described in mammalian cells: Trxl is a cytosolic protein with many different functions most of them depending on the redox active site (Holm- gren, 1985) and Trx2 is a mitocondrial protein whose only function so far described is to act as a electron donor for a mitochondria1 thiore- doxin-dependent peroxidase, thus being a major antioxidant defense in this organella (Spyrou et al.,

* To whom correspondence should be addressed: Dept. of Biosciences, Center for Biotechnology, Karolinska Institutet, Novum, 5-141 57 Huddinge, Sweden. Tel.: 46-8-6089162 ; Fax: 46-8-7745538 ; Email: giannis.spyrou@cbt.ki.se

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HUMAN THIOREDOXIN-LIKE PROTEIN GENE (TXL) 42 I

ATG TAA I I

- E l E2 E3 E4 E5 E6 E7 E8

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

TABLE 1 lntron/exon junction of human txl

Exon size Intron size Sequence a t exonlintron junction AA at piictioii ~ ~~

Exoiz (bpi liitron (bpi 5' splice donor 3 ' splice acceptor AA Position Type

1 303

2 97 1 11.885 AGAGGgtgag aacagGTGTG G 33 2

3 174 2 1.899 GTCAGgtaag tctagGGAAC G/G 65/66 0

4 123 3 6.161 GCTATgtatg ttcagATGGA Y/M 123/124 0

5 70 4 1.689 AACAGgtaat cacagCTGCT Q / L 1641165 0

6 173 5 1.648 TAATGgtgag tatagGTCAG G 188 1

7 105 6 3.326 TAACTgtaag ggtagATATT TI1 2451246 0

8 233 7 7.936 AACGAgtaag tctagGTAGT R / V 2801281 0

Exon sequences are in uppercase and intron sequences in lowercase. GT/ AG junctions are in bold. Txl cDNA sequence was used to determine the type of intron as well as position of the amino acids (Miranda-Vizuete et nl., 1998).

To assign the chromosomal localization of txl gene we searched the STS (sequenced tagged sites) deposited at the GenBankTM against txl- cDNA and 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 mark- ers are located on chromosome 18 at 71-83 centi- morgans 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 exten-

sively studied because its involvement in differ- ent 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 overexpres- sion, also maps in the same region (Ohno et al., 1987). Thioredoxin has been involved in tumor progression as it has been found to be overex- pressed in many human cancers and stimulates the proliferation of a wide variety of human solid tumors and leukemic cancer cell lines. Fur- thermore, a dominant-negative thioredoxin with its active site mutated is able to revert the trans- formed phenotype of human breast cancer thus

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422 ANTONIO MIRANDA-VIZUETE and GIANNIS SPYROU

Chr 18

P

11.3

11.2

11.1

11

12

21

4

22

23

H Mal ic enzyme 2, (ME2) TGFbeta-regulated MAD r e l a t e d protein,(MADRZ) Thioredoxin- l ike p r o t e i n ( t x l ) Squamous c e l l carcinoma antigen, (SCC) BCL-2 Ferrochelatase (FECH)

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

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 apop- tosis (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 signal-regulating kinase, and depend- ent of the status of its redox active site (Saitoh et al., 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 Dro- sophila database (A1402225 and AI402230) that when translated, resulted in a similar protein to that of human txl (accession number AF143404). Similarly, the search in a Caenorhabditis data- base 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

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HUMAN THIOREDOXIN-LIKE PROTEIN GENE (TXL) 423

FIGURE 3 Alignment of predicted amino acid sequence of human txl with the putative D. riirlni~og~zstrv and C. eleguuns homologues and human Trxl. 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

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, sepa- rated by two exons of 1912 and 871 by and both conforming the GT/AG rule. A comparison of the D. rnelanogaster and C. elegans txl together with human txl and Trxl is shown in Figure3. From the comparison of all these proteins, a very interesting pattern emerges as Drosophila and Caenorhabditis txl thioredoxin domains are much closer to human Trxl than human txl. Indeed, critical conserved residues for optimal thiore- doxin activity are absent in the human txl pro- tein while they are present in the others txl members (Figure 3). For instance, Asp26 (all numbering referred to human Trxl) which par- ticipates 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. 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 thi- olate 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 impor- tance to purify 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 cas-

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424 ANTONIO MIRANDA-VIZUETE and GIANNIS SPYROU

pases 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 quest ions.

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).

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