Identification of the first human glutaredoxin
pseudogene localized to human chromosome
20q11.2
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: Giannis.Spyrou@cbt.ki.se
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Database accession No.: AF227512
2
Thiol-disulfide interchange reactions are one of the most important regulatory
systems in cells. Two kinds of molecules are responsible for this process: non-protein
low molecular weight thiols and thiol-containing proteins of higher molecular weight
(Zlieger, 1985). Among the first type, glutathione arises as the most important
reductant in cell, while thioredoxin (Trx), glutaredoxin (Grx) and protein disulfide
isomerase (PDI) are the best known examples of regulatory proteins by thiol-disulfide
interchange reactions (Holmgren, 1989). Thioredoxins and glutaredoxins share many
common features like being small, heat-stable, globular proteins (around 12 kDa), with
a similar tridimensional structure (thioredoxin fold) and both using NADPH as source
of reducing equivalents (Holmgren, 1989). However, while the electron from NADPH
is transferred to the flavoenzyme thioredoxin reductase that in turn reduces
thioredoxin (the so-called thioredoxin system), glutaredoxin is reduced by the
sequential transfer of reducing power from NADPH to glutathione reductase and
glutathione (the so-called glutaredoxin system) (Holmgren, 1989). Once reduced, Trx
and Grx can act as general disulfide oxidoreductases with preference shown by Trx for
peptide substrates while Grx shows preference for low-molecular weight dithiolcontaining molecules (Holmgren, 1989). Grx was initially discovered as an alternative
hydrogen donor for the essential enzyme ribonucleotide reductase in a thioredoxindeficient mutant of E. coli (Holmgren, 1976; Holmgren, 1979). Since then Grx has also
been shown to be an electron donor for enzymes like adenosine 3´-phosphate-5´phosphosulfate reductase and methionine sulfoxide reductase, functions that Trx also
displays (Holmgren, 1989). In addition, Grx has been implicated in deiodination of
thyroxine to triiodothyronine and has shown dehydroascorbate reductase activity that
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generates ascorbic acid which protects neutrophils against the deleterious effects of the
respiratory burst (Goswami and Rosenberg, 1985; Park and Levine, 1996). More
recently Grx has been shown to regulate the DNA binding activity of the transcription
factor PEBP2 (Nakamura et al., 1999) and it has also been reported to play a role in
HIV-1 infection (Davis et al., 1997). Glutaredoxins have been isolated from all the
organisms studied and the common hallmark of the family is the conserved sequence
of the active site Cys-Pro-Tyr-Cys, except for pig Grx where tyrosine is changed to
phenylalanine. The human grx gene has been mapped to chromosome 5q14 and found
to be organized in three exons separated by 1.0 and 2.2 kb introns respectively (Padilla
et al., 1996; Park and Levine, 1997). However, two different mRNAs for human
glutaredoxin have been reported: a shorter form of 837 nucleotides, organized in 63
bases of 5´-UTR, 320 bases of ORF and 454 bases of 3´-UTR (Padilla et al., 1995) while
another larger form of 1328 nucleotides, identical to the shorter one except that it
contains an insertion of 566 nucleotides in the 3´-UTR (Park and Levine, 1996).
Surprisingly, this insertion is right at the position of the second intron junction,
separating the second and third exon but the intron length has been reported of 2.2 kb
while the insertion is only of 566 nucleotides (Park and Levine, 1997). Therefore, it is
reasonable to think that the second intron could indeed be composed of two introns
and one exon and thus Grx mRNA might be organized in four exons separated by
three introns. Thus, the Grx mRNA form isolated by Park and Levine (Park and
Levine, 1996) could be an alternative splicing variant expressing exon 3 while the form
described by Padilla et al. does not express such an exon (Padilla et al., 1995). Further
support for this hypothesis comes from the analysis of the expressed sequence tags
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(ESTs) available in the public databases. When EST databases were screened using the
larger Grx mRNA sequence, six EST overlapped different parts of the tentative third
exon thus demonstrating that this insertion is indeed expressed and might be
considered as an alternative splicing variant. The expression of this variant is much
lower that the shorter form described by Padilla et al. as seen by the higher number of
EST entries that do not contain the tentative third exon (Padilla et al., 1995). The
availability of the intron sequence described by Park and Levine would clarify this
point. We report here the identification of the first human glutaredoxin pseudogene
(Grx) that correspond to an inactive copy of the longer form of human Grx mRNA
described by Park and Levine (Park and Levine, 1996) and its localization at human
chromosome 20q11.2.
We commenced this study identifying an EMBL entry (HSBA425M5)
corresponding to a human genomic region of chromosome 20 that displayed high
homology with the human Grx mRNA described by Park and Levine (Park and
Levine, 1996). To further confirm this sequence we designed specific primers at the
flanking
regions
of
the
homology
GCACAATACCCCAAGCAATG-3´
and
interval
(Forward
Reverse
5´5´-
CGTTCTCACTAGAGACTGAAGAAGG-3´) and amplified by PCR a fragment from
human genomic DNA (Clontech). The amplified fragment was cloned into the pGEMTeasy vector (Promega) and sequenced in both directions confirming the sequence of
the EMBL entry HSBA425M5. As shown in Figure 1, the genomic fragment of clone
HSBA425M5 (from now named Grx) is highly homologous to human Grx mRNA,
including the 3´-UTR insertion reported by Park and Levine (Park and Levine, 1996).
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A comparison of Grx sequence with the two forms of Grx mRNA strongly suggests
that this genomic fragment might correspond to a processed pseudogene rather than a
functional copy of the grx gene that could have originated from a RNA intermediate
and integrated by retrotransposition in a different location in the genome (Vanin,
1985). For instance, Grx sequence contains multiple nucleotide changes when
compared to Grx mRNA that affect not only both UTRs but also the ORF. The most
striking changes are a stop codon shortly after the ATG start codon, a 12 bases
insertion that incorporates three extra valine residues although does not introduce any
frameshift and finally a one-base insertion that generates a frameshift resulting in a
different C-terminus of the protein including some in-frame stop codons (Figure 1).
The C-terminus of Grx contains cysteine residues that are required for enzymatic
activity as well as the glutathione binding site (Sun et al., 1998). These residues are not
present in the putative protein encoded by Grx, making unlikely that it would be
functional. In addition, the promoter region described for human Grx has been
replaced in Grx sequence as the homology with the mRNA ceases right after the
transcription start point (Park and Levine, 1997), the corresponding TATA and
CCAAT boxes have been replaced by an insertion of many copies of the sequence
repeat GAAG (Figure 1) and the two introns described for the human grx gene are not
present in Grx sequence. Furthermore, an imperfect polyA tail is present exactly
3´after the point at which the homology with the mRNA ends. Nevertheless, one of the
features of processed pseudogenes, the flanking direct repeats (Vanin, 1985), believed
to drive the retrotransposition event, are absent most probably due to the insertion of
the GAAG repeat sequence mentioned above.
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To further characterize this putative grx pseudogene we searched the sequence
tagged sites (STS) database using the whole HSBA425M5 clone sequence and
identified several STS sequences (G22160, G07631, G32972, G15102 and G19955) all of
them mapping between the gene markers D20S106 and D20S107 by PCR screening of a
human-rodent radiation hybrid panel. These gene markers are located on human
chromosome 20 at 50-55 centimorgans from the top of the linkage group. We
compared this location with the locations of other genes that have been mapped with
both fluorescence in situ hybridization and PCR screening of the same radiation
hybrid panel and show that the position of the Grx pseudogene can be assigned to
chromosome 20q11.2 (Figure 2).
Human glutaredoxin gene maps at chromosome 5q14 and two more bands at
chromosomes 12 and 14 hybridize with a human Grx probe in Southern blots
suggesting the presence of either novel active Grx isoforms or inactive pseudogenes
(Padilla et al., 1996). We report here the identification of the first human glutaredoxin
pseudogene, mapping at chromosome 20q11.2 which is in agreement with the fact that
processed pseudogenes are usually not syntenic with their respective functional genes
(Vanin, 1985).
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ACKNOWLEDGEMENTS
This work was supported by grants from the Swedish Medical Research
Council (Project 13X-10370), the TMR Marie Curie Research Training Grants
(contract ERBFMBICT972824) and the Karolinska Institutet Stiftelser.
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LEGENDS TO THE FIGURES
Figure 1. Comparison of the nucleotide sequence between human Grx
mRNA long (L) and short (S) forms and human Grx. The residues that differ
between Grx mRNA and Grx are marked with a triangle. Start and stop codons
are indicated by asterisks and the in-frame stop codon, the 12 bp insertion and the
1 base frameshift are boxed.
Figure 2. Chromosomal localization of human Grx. Human Grx is
located at 50-55 centimorgans from the top of the linkage group of chromosome 20.
Other genes mapping at the same region are also shown.
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