(Received for publication, April 6, 1995; and in revised form, June 26, 1995)
From the
Selenocysteine is co-translationally incorporated into
prokaryotic and eukaryotic selenoproteins at in-frame UGA codons.
However, the only component of the eukaryotic selenocysteine
incorporation machinery identified to date is the
selenocysteine-specific tRNA. In prokaryotes,
selenocysteine is synthesized from seryl-tRNA
and the
active selenium donor, selenophosphate. Selenophosphate is synthesized
from selenide and ATP by the selD gene product,
selenophosphate synthetase, and is required for selenocysteine
synthesis and incorporation into bacterial selenoproteins. We have now
cloned human selD and shown that transfection of the human selD cDNA into mammalian cells results in increased selenium
labeling of a mammalian selenoprotein, type 1 iodothyronine deiodinase.
Despite significant differences between the mechanisms of selenoprotein
synthesis in prokaryotes and eukaryotes, human selD weakly
complements a bacterial selD mutation, partially restoring
selenium incorporation into bacterial selenoproteins. Human
selenophosphate synthetase has only 32% homology with the bacterial
protein, although a highly homologous region that has similarity to a
consensus ATP/GTP binding domain has been identified. Point mutations
within this region result in decreased incorporation of selenium into
type 1 iodothyronine deiodinase in all but one case. Further analysis
revealed that reduced selenium labeling was due to altered ATP binding
properties of the mutant selenophosphate synthetases.
Selenocysteine is co-translationally incorporated into
prokaryotic selenoproteins at UGA codons(1) . It has been shown
recently that translation of the selenocysteine UGA codon in the
bacterial selenoenzyme, formate dehydrogenase, requires a specific
stem-loop in the coding region immediately 3` to the UGA
codon(2) . In addition, four genes, selA, selB, selC, and selD, are required for
selenocysteine codon recognition and
translation(3, 4, 5) . The selC gene product
is a selenocysteine-specific tRNA species that becomes charged with L-serine by seryl-tRNA synthetase(6) . The product of
the selA gene, selenocysteine synthase, converts
seryl-tRNA to selenocysteyl-tRNA
via an
aminoacrylyl intermediate(7, 8) . The active selenium
donor species in this reaction is selenophosphate, which is synthesized
by the selD gene product, selenophosphate
synthetase(9, 10) . In the final step of bacterial
selenoprotein synthesis, selenocysteyl-tRNA
is bound by a
specific translation factor, SELB, which also binds the stem-loop
structure, and decodes the UGA specifying
selenocysteine(11, 12) . Selenophosphate is also
required for conversion of 5-methylaminomethyl-2-thiouridine residues
in the anticodons of certain bacterial tRNAs to
5-methylaminomethyl-2-selenouridine(5, 6, 13, 14, 15) .
Several eukaryotic selenoproteins have also been identified, including glutathione peroxidases(16) , selenoprotein P(17, 18) , and the type I iodothyronine 5`-deiodinase(19) , all of which contain selenocysteine encoded by UGA codons. However, the mechanisms of recognition and translation of selenocysteine codons differ between prokaryotes and eukaryotes. Although stable stem-loop structures are required for eukaryotic selenoproteins synthesis, these elements are situated within the 3`-untranslated region of the mRNAs(20) . In addition, the sequences of prokaryotic and eukaryotic stem-loop structures are not conserved(2, 20) . Mutagenesis studies have also indicated that the position of the stem-loop in bacterial formate dehydrogenase is essential to function(2) , whereas in 5`-deiodinase, the position of the stem-loop is quite flexible(21) . Finally, cloning of the rat and human selenoprotein P cDNAs revealed the presence of 10 in-frame UGA codons (17, 18) and two 3`-untranslated stem-loops(21) . As yet, no prokaryotic seleno protein containing multiple selenocysteine residues has been identified.
Apart from the tRNA(22, 23) , none of
the components of the eukaryotic selenocysteine incorporation machinery
have been identified. This may be explained by poor conservation of the
selenoprotein translational machinery. For example, eukaryotic
selenoprotein mRNAs are unable to direct selenocysteine incorporation
when expression is attempted in Escherichia coli. (
)In addition, cloning of the tRNA
from
several organisms has revealed evolutionary differences in the
sequences for this tRNA within both the eukaryotic (23) and
the prokaryotic (24) kingdoms.
We have now cloned the human homologue of bacterial selD and shown that it is functional in mammalian cells and also in bacteria. Comparison of the human and bacterial selenophosphate synthetase peptide sequences reveals a high homology glycine-rich sequence that is similar to conserved sequences found in many ATP/GTP binding proteins and protein kinases(25, 26, 27, 28) . Mutational analysis has revealed that conserved amino acids within this region are important for selenophosphate synthetase activity and for the ATP binding properties of the enzyme.
Single colonies of BL21(DE3),
BL21(DE3)/pET-selD2, WL400-P3, WL400-P3/selD4, and
WL400-P3/pSL21 were grown in LB with appropriate antibiotics until the
cells were in log phase growth. Cultures were then diluted 1:4 in LB
and 6 µCi of Se was added to each. 4 mM
isopropyl-
-D-thiogalactopyranosidase was added to both
BL21(DE3) cultures to induce the T7 RNA polymerase gene and
consequently transcription of the selD plasmid.
Se labeling was continued for 3 h before bacterial pellets
were recovered, resuspended in distilled water, and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography.
Time course experiments with
8-azido-[-
P]ATP were also carried out to
examine potential differences between selD4 and the selD mutants. Reaction mixtures were assembled as described above and
allowed to incubate on ice for varying lengths of time before UV
cross-linking, then reactions were stopped by the addition of excess
dithiothreitol. Labeled proteins were analyzed by polyacrylamide SDS
gel electrophoresis and autoradiography.
The deduced amino acid sequence of human selD is 381 amino acids with a calculated molecular mass of approximately 45 kDa and shows overall 32% identity and 55% similarity with E. coliselD. Alignment of the human and E. coli peptide sequences (Fig. 1) revealed a conserved glycine rich region with the sequence Gly-Phe-Gly-Ile-Leu-Gly-His at 269-275 in the human peptide, and at 232-238 in the E. coli sequence (Fig. 1). This sequence is similar to the conserved ATP/GTP binding consensus sequences, Gly-X-X-X-X-Gly-Lys-Ser/Thr and Gly-X-Gly-X-X-Gly found in many ATP/GTP binding proteins and protein kinases, respectively(26, 27, 28, 29) .
Figure 1: Alignment of the predicted amino acid sequences of human and E. coli selenophosphate synthetases. Uppersequence is human selD, and lowersequence is E. coliselD. Conserved amino acids between the human and bacterial enzymes are indicated by verticallines. The glycine-rich, putative ATP binding consensus sequence is indicated by an asterisk; methionine residues in both sequences are indicated by +.
Figure 2:
Expression of selenophosphate synthetase
in mammalian cells. HtTA-1 cells were transfected with human selD (selD4; 10 µg/dish), E. coliselD (SL21; 10 µg/dish), XtRNA (encoding the Xenopus tRNA; 5 µg/dish), and G21-D10
(encoding rat 5`-deiodinase (5`DI) 5 µg/dish) as
indicated. Cells were harvested following transfection and 18 h in
vivo labeling with 6 µCi of
Se. Proteins were
then analyzed on 10% polyacrylamide SDS gels followed by
autoradiography.
Complementation of a Bacterial
selD Mutation-E. coli strain BL21(DE3) contains
endogenous selD, thus in vivo labeling with Se results in the incorporation of selenium into formate
dehydrogenase and 5-methylaminomethyl-2-selenouridine residues in tRNAs (Fig. 3). Interestingly, when BL21(DE3) was transformed with
pET-selD2, selenophosphate synthetase itself became labeled
with
Se. In contrast, in WL400-P3, which contains an
interrupted and therefore inactive selD gene(5) ,
neither the tRNAs nor formate dehydrogenase are labeled with
Se, confirming that an active selD gene is
required for selenium incorporation into bacterial selenoproteins and
tRNAs (Fig. 3).
Se labeling of formate
dehydrogenase and tRNAs is restored when WL400-P3 are transformed with
an intact E. coliselD gene (pSL21) or the intact
human selD cDNA (selD4). However, the levels of
incorporation of
Se into formate dehydrogenase and tRNAs
in the presence of selD4 are much lower than in bacteria
transformed with E. coliselD (Fig. 3).
Figure 3:
Complementation of a bacterial selD mutation. E. coli strain WL400-P3 containing an
inactivated selD gene was transformed with either bacterial (SL21) or human (selD4) selD as indicated.
Controls included WL400-P3 alone, the wild-type E. coli strain
BL21(DE3), which contains endogenous prokaryotic selD, and
BL21(DE3) transformed by human selD in a bacterial expression
vector (pET selD). Bacteria were labeled in vivo with Se for 3 h. Proteins were analyzed on 10% polyacrylamide
SDS gels followed by autoradiography.
Figure 4:
Expression of selD mutants in
mammalian cells. HtTA-1 cells were transfected with 10 µg of either
wild type selD (selD4) or selD mutants (10
µg) as indicated. Cells were simultaneously transfected with
G21-D10 (expressing 5`-deiodinase (5`DI); 5 µg) and XtRNA
(encoding the Xenopus tRNA; 5 µg). Following
transfection, cells were labeled with
Se for 18 h, and
proteins were analyzed on 10% polyacrylamide SDS
gels.
Figure 5:
8-azido-[-
P]ATP
binding properties of selD. HtTA-1 cells were transfected with
wild-type human selD (selD4; 10 µg/dish) or selD mutants (10 µg/dish) as indicated. Photoaffinity
labeling of cell extracts with
8-azido-[
-
P]ATP was carried out as
described under ``Experimental Procedures.'' ATP binding was
analyzed on 12% polyacrylamide SDS gels followed by
autoradiography.
Following ATP binding to selenophosphate synthetase, ATP is
hydrolyzed to produce AMP and a pyrophosphate-enzyme
intermediate(9, 10) . In order to examine ATP
hydrolysis by wild-type human and mutant selenophosphate synthetases,
cell extracts were incubated with ATP, and the reaction products were
analyzed by thin-layer chromatography. Data from this study indicated
that the wild-type and mutant enzymes do hydrolyze ATP (data not
shown). However cell extracts alone also extensively degrade ATP, and
thus rates of hydrolysis by the selenophosphate synthetases were
difficult to measure accurately. Therefore we have examined the time
course of ATP binding. Wild-type selD4 initially exhibits
efficient ATP binding, followed by a rapid loss of bound label (Fig. 6). In contrast, the amount of ATP binding by the
His
Tyr mutant decreases gradually over the same
time period, suggesting less ATP hydrolysis by this mutant (Fig. 6). Gly
Cys and Gly
Arg also bind more ATP for a longer period of time than
wild-type selD (Table 1, and data not shown) suggesting
that glycine residues at position 269 and 271 may be involved in ATP
hydrolysis (Table 1). However, due to degradation of ATP by cell
extracts, definitive information on ATP hydrolysis by wild-type human selD and the mutants will require purification of the enzymes.
Figure 6:
ATP hydrolysis properties of selD. Extracts of HtTA-1 cells transfected with either
wild-type selD (selD4; 10 µg/dish) or selD mutants (10 µg/dish) were incubated in the presence of
8-azido-[-
P]ATP for various time periods
before being cross-linked under ultraviolet radiation for 3 min. The
time course of ATP binding was analyzed on 12% polyacrylamide SDS gels
followed by autoradiography.
The role of prokaryotic selenophosphate synthetase in the production of the active selenium donor, selenophosphate, has been clearly demonstrated(3, 4, 5, 13, 14, 15) . Mammalian selenoproteins have also been identified(16, 17, 18, 19, 20) , although the mechanism whereby selenocysteine is synthesized and incorporated into these proteins is not yet clear. We have now cloned the human homologue of bacterial selD and shown that it is functional in both mammalian cells and bacteria. A partial clone of human selD was obtained using a yeast two-hybrid protein interaction system. The bait protein for this interaction was the mammalian spliceosome-associated protein SAP62, which is involved in mRNA splicing(42) . Since appropriate control experiments were performed to minimize nonspecific interactions with the bait protein, the interaction between selD and SAP62 may be specific, although the relevance of this and the implication that selD may be involved in RNA splicing is not known.
Transfection of E. coli and human selD into mammalian cells showed
that both prokaryotic and eukaryotic selenophosphate synthetases are
functional in higher eukaryotes. However, when human selD was
transformed into bacteria containing an inactive selD gene, it
only partially complemented the bacterial system and produced low
levels of Se labeled tRNAs and formate dehydrogenase. One
possible explanation for this may be that the human selD gene
product is improperly folded in bacteria. Alternatively, a factor(s)
found in mammalian cells but not bacteria may be required in addition
to the mammalian selD gene product to maintain normal
selenophosphate synthetase activity. Nevertheless, these data do
indicate that although human and bacterial selenophosphate synthetase
sequences are poorly conserved (32% homology), and there is a great
evolutionary distance separating the two proteins, the action of selD is probably very similar in bacteria and higher
organisms. When the E. coli strain BL21(DE3) was transformed
with human selD and grown in the presence of
Se,
in addition to the expected bacterial selenoproteins becoming labeled,
selenophosphate synthetase itself was also labeled by
Se (Fig. 3). This was unexpected, since the sequence of human selD does not indicate the presence of selenocysteine within
the protein itself. However, when the selenium to sulfur ratio is
higher than normal, selenium may be nonspecifically misincorporated
into proteins in the form of
selenomethionine(43, 44) , or, if the methionine
content of a protein is high, misincorporation of selenium into
selenomethionine may also occur(45, 46) . It was
therefore interesting to note that human selenophosphate synthetase
contains 18 methionine residues, and the bacterial protein contains 13,
both of which are much higher than the average protein methionine
content and may therefore explain the observed labeling of selD.
Mutational analysis of the putative ATP binding
consensus sequence in the human selD cDNA indicated that all
of the amino acids considered are important in normal selenophosphate
synthetase activity. We have also shown that the residues within this
region are important for normal ATP binding properties of
selenophosphate synthetase, although direct measurement of ATP
hydrolysis will require purification of wild-type and mutated enzymes.
Recent work on bacterial selD involved mutational analysis of
the sequence encoding the amino terminus of the protein, which also
shared some similarity with the consensus ATP binding
sequence(47, 48) . It was shown that the cysteine
residue at position 17 (Cys) and the lysine residue at
position 20 (Lys
) are both essential for the formation of
selenophosphate from selenide and ATP(47, 48) , while
Cys
was also shown to be required for ATP binding to
selenophosphate synthetase. In contrast, mutation of Cys
had no effect on selenophosphate activity. The significance of
these results in relation to human selenophosphate synthetase activity
is not clear since the only residue in this region that is conserved
between the two sequences is Cys
, which was not essential
for catalytic activity in E. coliselD. However it is
possible that there are differences in the three-dimensional structure
of the bacterial versus human selD gene products,
which could explain the discrepancies between the two studies. Further
work is clearly required to determine the importance of individual
amino acids in both selD homologues for normal selenophosphate
synthetase function, ATP binding, and ATP hydrolysis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34044[GenBank].