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INTRODUCTION |
Free ADP-ribose (ADPR)1
is a potentially toxic metabolite (reviewed in Ref. 3), and in addition
has recently been demonstrated to act as a gating molecule for the
TRPM2 ion channel (1), suggesting that it may have a signaling function
in certain contexts. Production of free ADP-ribose occurs via a variety
of pathways, and it is not presently clear which of these is most
important in vivo. However, the catabolism of free
ADP-ribose is thought to occur via a single pathway, hydrolysis to
AMP and ribose-5-phosphate via ADP-ribose pyrophosphatases
(ADPRases) (4-7). In humans, four distinct ADPRase activities have
been described including three cytosolic activities (ADPRase-I,
-II, and -Mn) and the mitochondrial ADPRase-m (4, 6, 7) based on
traditional protein purification approaches. In contrast, cDNA
cloning and characterization has identified only two human proteins,
designated NUDT5 and NUDT9, respectively, having ADPRase activity (1,
8). NUDT5 is a cytosolic enzyme catalyzing the hydrolysis of ADP-ribose
and other ADP-sugar conjugates (8). It appears to correspond to the
previously described ADPRase-II based on the similarities of their
reported substrate ranges. To date, NUDT9 has been shown to be highly
specific for ADP-ribose, but has otherwise not been characterized in
detail (1).
Here, we report the characterization of the human NUDT9 gene and its
potential for alternative splicing and results from analyses of the
physical, enzymatic, and cell biological properties of human NUDT9. Our
results indicate that NUDT9
represents the previously characterized
ADP-ribose-m, and provide insight into NUDT9 structure/function relationships as well as the relationship between NUDT9 proteins and a
highly homologous domain (NUDT9H) of the TRPM2 calcium-permeable cation channel.
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MATERIALS AND METHODS |
cDNA Cloning and Sequence Analyses--
Human cDNA
transcripts were isolated as previously described from a spleen
cDNA library (1). BLAST alignments were performed using the NCBI
BLAST server. Computer analysis of sorting signals was performed using
the PSORT and iPSORT algorithms available at the GenomeNet site in
Japan. All other analyses and ClustalW alignments were performed using
the MacVector program (Oxford Biotechnology). Clustal tree analysis was
calculated using the nearest neighbor method with an open gap penalty
of 10.0, extend gap penalty of 0.1, delay divergent of 40%, gap
distance of 8, and a Block's substitution matrix similarity matrix.
Analysis of the human NUDT9 genomic structure was performed by aligning
the NUDT9 mRNA sequence with the working draft of chromosome 4 available from GenBankTM and comparing the aligning regions
with the predicted exons from the annotated version of chromosome 4 (exons were predicted by Genomescan). The exons based on alignment
closely corresponded to those in the annotated sequences, with the
primary exception that the first exon based on alignment starts 20 base
pairs downstream of that predicted by Genomescan. Based on these
approaches, the actual exons and corresponding bases in NUDT9 would be
assigned as: exon1 = 6942506-6942100 (base pairs 26-432 of the
NUDT9 mRNA), exon 2 = 6930131-6929892 (base pairs 433-672 of
the NUDT9 mRNA), exon3 = 6925969-6925874 (base pairs 673-768
of the NUDT9 mRNA), exon4 = 6922417-6922331 (base pairs
769-855 of the NUDT9 mRNA), exon5 = 6915104-6914993 (base
pairs 856-965 of the NUDT9 mRNA), exon6 = 6912657-6912511
(base pairs 966-1114 of the NUDT9 mRNA), exon7 = 6909875-6909791 (base pairs 1115-1199 of the NUDT9 mRNA), and
exon8 = 6906403-6905901 (base pairs 1200-1704 of the NUDT9 mRNA). Genomic base pair designations are for Homo sapiens
chromosome 4 working draft sequence segment NT_006204.11 Hs4_6361.
Construction of Escherichia coli Expression Constructs for
NUDT9
and NUDT9-H Region of TRPM2--
Full-length coding sequences
for NUDT9
and NUDT9
were produced by PCR to place an
NcoI site at the 5' end of the coding sequence and a
NotI site at the 3' end, and subcloned into the pET-24d T7
expression vector (Novagen). For the TRPM2 NUDT9 homology region, a
construct was made by PCR to include a BspHI site, an artificial start codon, amino acids 1197-1503, a stop codon, and a 3'
NotI site. This was also subcloned into pET-24d. Other
mutant forms of NUDT9 (see figure legends) were constructed using
site-directed mutagenesis (QuikChange, Stratagene) of the NUDT9/pET-24d
construct. Correct sequences of all constructs were confirmed by DNA sequencing.
E. coli Expression and Purification of NUDT9 and the NUDT9
Homology Region of TRPM2 and Assays for Nudix-type Activity of NUDT9
and NUDT9-H Region of TRPM2--
Performed as previously described
(1).
Analytical Ultracentrifugation--
Analytical
ultracentrifugation experiments were carried out on a Beckmann
Instruments Optima XL-A with absorbance optics. Protein solutions of
A280 = 0.21-0.42 (corresponding to a
concentration of 3-6 µM in terms of protein monomer)
were centrifuged at 20 C°. The buffer solution contained 50 mM Tris-HCl, pH 7.5, 50 mM KCl, and 0.1 mM dithiothreitol. The effect of Mg2+ was
studied by supplementing the buffer with MgCl2 to a
concentration of 5 mM. For the NudT9 protein the following
parameters were derived with the program SEDNTERP version 1.05 (J. Philo, D. Hayes, T. Laue; freely downloadable at
alpha.bbri.org/rasmb/spin/ms_dos/) from the amino acid sequence:
molecular mass of Mr = 36.2 kDa, extinction
coefficient at 280 nm
280 = 67050 M
1·cm
1, partial specific
volume
= 0.728 ml·g
1 at 20 °C,
and a hydration of 0.44 g H2O/g protein. The same
program was used to calculate the buffer density and viscosity at
20 °C yielding
= 1.0020 g·ml
1 and
= 1.0124 milli-Pascal sec (buffer without MgCl2) or
= 1.0024 g·ml
1 and
= 1.0126 milli-Pascal sec (buffer with 5 mM MgCl2), and for modeling the apparent dimensions of an oblate or prolate including hydration according to the Teller method. Sedimentation equilibrium runs were conducted at 10,000 and 25,000 rpm. Equilibrium was reached
after about 20 h of centrifugation as judged from comparison of
successive scans. The baseline offset between sample and buffer was
determined from the absorbance of the region close to the meniscus
after sedimenting the protein sample at 48,000 rpm for 6 h.
Equilibrium data were evaluated by fitting to a single exponential function as described previously (9). The sedimentation velocity data
were recorded at 42,000 rpm using a spacing of 0.01 cm with four
averages in the continuous scan mode. Data were analyzed by computing
the sedimentation coefficient distribution (g(s*)) distribution (10, 11) with the program DCDT+ version 1.13 by John Philo
according to the algorithm described (12).
Partial Proteolysis of Nudt9 and Subsequent Analysis of the
Obtained Fragments--
A serial dilution from a 1 mg/ml stock
solution of the nonspecific cysteine protease bromelain (Calbiochem)
was made, and the appropriate protease concentration for the partial
proteolysis of purified Nudt9 (0.75 mg/ml final) was determined. A
final bromelain concentration between 0.75 and 6.25 µg/ml in the
protease reaction (60' incubation at 37 °C) gave a clear and
reproducible pattern showing one main subfragment around 20 kDa. The
time course analysis of the protease reaction was performed using a
1.56 µg/ml final concentration of bromelain. The reaction mixture
contained beside the proteins, 25 mM Tris, pH 7.5, 100 mM NaCl, 1 mM MgCl2, and 1 mM dithiothreitol. The reactions were stopped by the
addition of a 10-fold excess of iodoacetamide followed by SDS-PAGE. Two stable fragments from the Coomassie-stained gel were subjected to
MALDI-TOF mass spectroscopic analysis in a Bruker Daltonic Spectrometer
(Billerica, MA) operated in the reflectron mode. Spectra were
externally calibrated with a standard peptide mixture and analyzed
using the program "m/z" from Proteometrics
(Winnipeg, Manitoba). Coomassie-stained gel bands were digested in-gel
with trypsin (omitting reduction and alyklation). Following overnight digestion, the digestion buffer was removed from the gel slices and
trifluoroacetic acid was added to a final concentration of 0.5%. A
5-µl fraction of the digestion mixture was concentrated and purified
using a nano-scale column and column elution was accomplished with the
matrix solution (0.1% trifluoroacetic acid/50% acetonitrile saturated
with alpha-cyano-4-hydroxycinammic acid) onto the MALDI target.
To determine the N-terminal residues of the obtained main fragment
the partial proteolysis reaction was separated by SDS-PAGE in a
12.5% polyacryalmaide gel and transferred to a polyvinylidene difluoride membrane (Millipore). The obtained protein bands were stained by Coomassie and excised for further analysis by
Erdman-degradation chemistry.
Eukaryotic Expression of NUDT9--
Wild type human NUDT9
and
NUDT9
cDNAs were subcloned to pCDNA4/T0, transfected into
either tet-supressor expressing DT-40 B-cells or HEK-293 cells by
electroporation, and zeocin-resistant clones were isolated. Cells
untreated with tetracycline showed no detectable protein expression,
whereas treated cells produced the level of protein expression
illustrated in Fig. 4A. A NUDT9
-EGFP fusion protein was
constructed by placing a KpnI site internal to the NUDT9
stop codon and ligating this to an EGFP (Clontech) construct containing a KpnI site placed just internal to the
start codon. This construct was ligated into the NotI and
XhoI sites of the pCDNA4/TO vector (Invitrogen). The
NUDT9
-EGFP in pCDNA4/T0 was transfected into HEK-293 cells and
DT-40 cells expressing the tet-repressor, and zeocin-resistant clones
were isolated. Cells untreated with tetracycline showed no detectable
protein expression, whereas treated cells were obviously fluorescent
and showed the staining patterns illustrated in Fig. 4B.
Protein Analysis--
Typically cells were lysed at 5 × 106 cells per ml of lysis buffer, and lysates were
cleared by centrifugation at 14,000 RPM for 15 min prior to preparation
for SDS-PAGE. SDS-PAGE and immunoblotting were performed using standard
techniques. Anti-NUDT9 antibody was generated by immunizing rabbits
with a peptide DDPRNTDNAWMETEAVNYHDETGE, corresponding to amino acids
270-293 near the C terminus of the enzyme.
Fluorescence Imaging--
Cells expressing NUDT9
-GFP and
NUDT9
-GFP fusion proteins were plated on cover slips and digitally
imaged at 40× magnification. The conditions used produced no
detectable fluorescence from uninduced cells. For co-localization
experiments, mitochondria and nuclei were separately stained with
Mito-Tracker dye and Hoechst 33342 (Molecular Probes), respectively.
Using conditions in which no bleed through was detected from
Mito-Tracker or Hoechst staining into GFP channels, images of
NUDT9
-GFP, Mito-Tracker, and Hoechst staining were separately
collected using filter sets specific for each dye, respectively.
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RESULTS |
Computer Analysis of the Human NUDT9 Gene--
The NUDT9 gene is
located on chromosome 4 as previously reported by Lin et al.
(2), who also reported the presence of two NUDT9 transcripts designated
NUDT9
and NUDT9
(nomenclature that we will adhere to for both the
transcripts and specific proteins for this report, whereas when
referring to enzymatic activity we will simply refer to NUDT9). Based
on comparison of the NUDT9
and NUDT9
cDNAs, Lin et
al. (2) suggested that the NUDT9 primary transcript is
alternatively spliced to produce these two transcripts via inclusion of
an intron in the longer NUDT9
transcript. However, their study did
not include an analysis of the NUDT9 genomic structure. We have
analyzed the structure of the human NUDT9 gene using sequences available from public databases (Fig. 1),
and our analysis indicates that the NUDT9 gene consists of 8 well
defined exons spread over ~37 kb. Based on this gene structure, the
NUDT9
variant transcript is not produced by removal of an intron but
is instead the result of a potentially aberrant splice from a splice
donor within the first NUDT9 exon to the splice acceptor site at the
beginning of exon 2. This conclusion is based on: 1) the presence of
the entire putative exon 1 as defined here in the reported NUDT9
transcript; 2) the presence of the entire putative exon1 as defined here as a contiguous sequence within the present chromosome 4 working
draft sequence; and 3) the presence of a large (>10 kb) well defined
intron adjacent to exon 1, which is spliced from both the NUDT9
and
NUDT9
transcripts. As a means of assessing the relative abundance of
NUDT9
and NUDT9
transcripts, we end-sequenced twenty-two separate
NUDT9 clones that had been isolated utilizing a selection cloning
method targeting sequences between base pairs 597 and 849 of the
NUDT9
transcript (thereby targeting sequences contained within exons
2 and 3, such that both NUDT9
and NUDT9
transcripts would be
isolated). Of these twenty-two clones, eighteen contained either
full-length or nearly full-length transcripts corresponding to
NUDT9
. Of the four transcripts with alternative 5' ends, two were
truncated within exon 1 after the start of the predicted NUDT9
coding sequence, one was truncated at the end of exon 2, and one
represented the previously reported NUDT9
transcript. These results
indicate that in human spleen, the tissue from which our NUDT9
sequences were cloned, the NUDT9
transcript is by far the dominantly
expressed transcript. Determining the question of whether there is any
physiological relevance of NUDT9
or if it simply represents a low
abundance aberrant transcript will require investigation into whether
there are proteins in the native tissue corresponding to the predicted
product of the NUDT9
transcript. This task will be complicated by
the fact that NUDT9
is extensively processed (see below) leading to
the production of proteins extremely close in size to that of the
protein predicted to be encoded by NUDT9
.

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Fig. 1.
Organization of the NUDT9 gene and splicing
of NUDT9 and NUDT9
transcripts. The light gray portion of exon 1 in
the splicing schematic for the NUDT9 transcript denotes the portion
that is spliced out of NUDT9 in addition to the large first intron.
The black box at base pair 70 of exon 1 denotes the apparent
cryptic splice donor site within exon 1.
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An alignment of the predicted NUDT9
protein sequences from several
organisms is shown in Fig. 2A.
As can be seen, NUDT9 is highly conserved, with >60% identity and
~80% similarity at the amino acid level. BLAST alignments of the
human, mouse, and chicken sequences with various NCBI databases
identified close homologs in Drosophila melanogaster
(droNUDT9 in Fig. 2A) and Caenorhabditis elegans (celNUDT9
in Fig. 2A) based on the presence of the conserved Nudix box
(black outline box) and highly conserved regions both upstream and downstream from it (dark gray outline boxes).
Interestingly, these analyses failed to identify significantly related
sequences in any prokaryote or unicellular eukaryote. Computer analysis of the N-terminal 100 amino acids of human NUDT9
using PSORT and
iPSORT had previously demonstrated the presence of a conserved putative
signal peptide or subcellular compartment targeting presequence (1).
Similar analyses of murine NUDT9
gave identical results. Although
the chicken NUDT9
sequence is not yet complete, it appears that the
signal peptide/targeting presequence is present in the chicken version
as well based on the degree of conservation of this region relative to
that of human and murine NUDT9
predicted proteins. This suggests
that vertebrate NUDT9 homologs are all likely to be either secreted or
subcellularly compartmentalized. Computer analyses of the N termini of
either droNUDT9 or celNUDT9 did not predict the presence of a signal
peptide or subcellular targeting presequence, although the degree of
aliphilicity of this region in both of these enzymes is suspicious for
their functioning in this manner as well.

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Fig. 2.
Alignment and phylogenetic analysis of NUDT9
homologs. A, alignment of various species of NUDT9 homologs.
Alignment was performed using ClustalW. and denote the
initiating methionines of the NUDT9 and NUDT9 proteins,
respectively. B, phylogenetic relationship of NUDT9 homologs
and four other examples of Nudix family ADPRases. The evolutionary tree
was calculated using the neighbor joining method. Evolutionary distance
is shown by the total branch length (horizontal
lines).
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The evolutionary relationship of the various NUDT9 homologs and four
other proteins with ADPRase activity, NUDT5, Saccharomyces cerevisiae ADPRase, E. coli ADPRase, and
Methanococcus jannaschii ADPRase, is summarized in a
CLUSTAL phylogenetic tree graph (Fig. 2B). Based on this
analysis, E. coli ADPRase and NUDT5 and the S. cerevisiae ADPRase appear to represent families distinct from a
well defined family containing the various NUDT9 homologs, consistent with the broader substrate specificities of these three enzymes (5, 8,
13). In contrast, the M. jannaschii ADPRase is grouped more
closely to the NUDT9 family, consistent with its high degree of
specificity for ADP-ribose and the similarity of its metal ion
requirements to those of NUDT9 (Ref. 14 and see below). Whether the
degree of similarity observed between the M. jannaschii (an
Archaeon) enzyme and the NUDT9 family is due to divergent or
convergent evolution is not presently clear. As more full organism
genomes become available for analysis, greater insight into the
evolutionary relationship of highly specific ADPRases may be possible.
Analysis of NUDT9
and NUDT9
Subcellular Localization--
As
discussed above and as illustrated in Fig. 2 (light gray outline
box), all vertebrate NUDT9 homologs have an apparent N-terminal signal peptide/targeting signal in their major transcripts (designated NUDT9
), which is potentially missing in the NUDT9
variant
transcript. To investigate what role this sequence might play during
NUDT9
biosynthesis, we transfected DT-40 B-cells or HEK-293 cells
with NUDT9
and NUDT9
cDNAs and analyzed NUDT9
expression
by immunoblotting with an anti-NUDT9 antibody (Fig.
3). NUDT9
transfection induced the
appearance in cell lysates (but not in the culture supernatants) of
three new bands in the 30-40 kDa molecular mass range (Fig. 3A, left panel, the small blips at 45 kDa in the uninduced lane appear to represent spurious signal
due to a slight defect in membrane blocking)). Identical results were
obtained in HEK-293 cells (data not shown). We interpret the abundance
of anti-NUDT9 immunoreactivity in cell lysates as most consistent with
the NUDT9
N-terminal region acting as a sorting signal for
subcellular compartmentalization as opposed to secretion, and the
presence of three bands as most consistent with proteolysis of both the
predicted NUDT9
signal peptide and a second sequence as well.
Consistent with its lack of a predicted signal sequence, NUDT9
transfection (Fig. 3B, left panel) produced only
a single NUDT9-immunoreactive band, presumably because it lacks the
sequence proteolyzed or because the product of the NUDT9
transcript
was not targeted to the compartment containing the requisite protease.
To directly determine where the NUDT9
and NUDT9
protein products
were subcellularly localized, we produced NUDT9
and NUDT9
C-terminal-EGFP fusion protein expression constructs and imaged their
subcellular localization. As shown in Fig. 3A (middle
panel), the NUDT9
-GFP fusion protein produced a
compartmentalized staining pattern consistent with its localization to
mitochondria or other large organelle in DT-40 B-cells. Because DT-40
cells have little cytoplasm, they are not an optimal system for
organellar marker co-localization studies. Therefore we used the same
NUDT9
-GFP expression construct to transfect HEK-293 cells and imaged
its subcellular localization in conjunction with specific staining of
mitochondria with Mito-Tracker dye and nuclei with Hoechst 33342 (Fig.
3A, right panel). As can be seen, the NUDT9
-GFP fusion protein is visible in structures with a morphology highly characteristic of mitochondria and that stain specifically with
the Mito-Tracker dye. The same pattern was observed when NUDT9
-GFP
was expressed and imaged in the absence of Mito-Tracker staining (data
not shown). In contrast (Fig. 3B, right panel), the NUDT9
C-terminal-EGFP fusion protein showed no apparent
subcellular localization when expressed alone. From the above studies,
we conclude that NUDT9
is produced as a precursor polypeptide and specifically imported into mitochondria where it undergoes a two step
processing to its mature form, whereas NUDT9
is neither specifically
compartmentalized nor further processed.

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Fig. 3.
Eukaryotic expression and subcellular
localization of NUDT9. A, eukaryotic expression of
NUDT9 . Left panel, a human NUDT9 expression construct
was transfected as described under "Materials and Methods" and
analyzed by SDS-PAGE and immunoblotting with an anti-NUDT9 antibody.
Middle panel, localization of NUDT9 -GFP in chicken DT-40
B-cells. Chicken DT-40 B-cells were transfected with a NUDT9 -GFP
fusion protein construct, and the subcellular localization of this
construct was imaged as described under "Materials and Methods."
Right panel, HEK-293 cells were transfected with a
NUDT9 -GFP fusion protein construct, and the subcellular localization
of this construct (green), cellular mitochondria
(red), and nuclei (blue) were imaged separately
as described under "Materials and Methods." B,
eukaryotic expression of NUDT9 . Left panel, a
human NUDT9 expression construct was transfected as described under
"Materials and Methods" and analyzed by SDS-PAGE and immunoblotting
with an anti-NUDT9 antibody. Right panel, HEK-293 cells were
transfected with a NUDT9 -GFP fusion protein construct, and
the subcellular localization of this construct was imaged as described
under "Materials and Methods."
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NUDT9 Association State Studied by Gel Permeation Chromatography
and Analytical Ultracentrifugation--
Many Nudix enzymes are dimers
(15, 16), and recent structural data on the E. coli ADPRase
indicate that it functions as a dimer (17), suggesting this might be
the case for other members of the classical ADPRase family. To
determine whether a similar mode of function applies to NUDT9, we
analyzed the molecular weight of purified enzymatically active human
NUDT9, which has a calculated molecular mass
(Mr) of 39,121 daltons, by gel
permeation chromatography and analytical ultracentrifugation. On gel
permeation chromatography, NUDT9 eluted as a single sharp peak at ~60
ml elution volume, corresponding to Mr
40 kDa based on our column calibration (Fig. 4A). The sharp
peak and close correlation of calculated molecular mass with the
molecular mass predicted from chromatographic behavior are strong
evidence that, under the conditions used, NUDT9 is entirely monomeric
in form.

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Fig. 4.
NUDT9 association state studied by gel
permeation chromatography and analytical ultracentrifugation.
A, gel permeation chromatagraphy of NUDT9. NUDT9 protein was
chromatographed on a Fractogel-EMD-Biosec column, and its elution
volume was measured as 58 ml. Five standards used for column
calibration (Vit B12, aprotinin, cytochrome C, carbonic anhydrase, and
bovine serum albumin) are plotted with NUDT9 on the right. A
regression line calculated using the known Mr of
the standards and their measured elution volumes predicts a molecular
mass of ~36 kDa for NUDT9. B, example of a sedimentation equilibrium run in
Mg2+-containing buffer of NUDT9 protein at a concentration
of 6 µM and a rotor speed of 10,000 rpm. In the
bottom part of the figure the measured absorbance at 280 nm
versus the radial position (distance to the center of the
rotor) is shown. The top part of the figure gives the
residuals to the fit expressed as the difference between experimental
and fitted values. Only every third data point is shown. For the data
presented in this figure a molecular mass of 38.0 kDa was determined.
All equilibrium data recorded at different concentrations and two rotor
speeds gave an average molecular mass of Mr = 38.9 ± 2.5 kDa. C, sedimentation velocity run of NUDT9
analyzed by the g(s*) method in
Mg2+-containing buffer. The fit curve corresponds to a
sedimentation coefficient of s20,w = 3.1 S
(maximum of the distribution) and a diffusion constant of
D20,w = 7.4·10 7 cm2
sec 1 determined from the width of the gaussian-shaped
function. The molecular mass calculated from s and
D in this experiment was Mr = 37 kDa.
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We next studied the association state and hydrodynamic shape of NUDT9
by analytical ultracentrifugation. Fig. 4B shows an example
of a sedimentation equilibrium run of NUDT9 protein, whereas Fig.
4C displays the analysis of a sedimentation velocity
experiment by the g(s*) method (10, 11). Both
runs were conducted in Mg2+-containing buffer. In the
absence of MgCl2, the sedimentation properties of the
proteins remained unchanged (data not shown). The average molecular
weight determined from the sedimentation equilibrium runs of multiple
samples at different protein concentrations and examined at two rotor
speeds was Mr = 38.9 ± 2.5 kDa. In the sedimentation velocity analysis a sedimentation coefficient of s20,w = 3.1 ± 0.1 S (maximum of the
distribution) and a diffusion constant of D20,w = (7.4 ± 0.5) ·10
7 cm2
sec
1 determined from the width of the distribution were
obtained. These values of s and D correspond to a
molecular mass of 37 ± 4 kDa as calculated according to the
Svedberg equation. Based on the above analyses, we conclude that, in
contrast to the bacterial ADPR pyrosphosphatase, NUDT9 is likely to be
active in monomeric form. With an estimated hydration of 0.44 g
H2O/g protein for NudT9 the ratio of the measured friction
coefficient f (calculated from s and
M) to that of a sphere with the same volume
f0 was f/f0 = 1.10. The corresponding axial ratios for the hydrodynamic dimensions of
the protein are 2.8 assuming a disk-shaped oblate (7.3 diameter and 2.6 nm height) and 2.7 for a rod-like prolate (10.1 length and 3.7 nm
diameter), indicating that the shape of NUDT9 in solution deviates
significantly from that of a sphere.
Metal Ion and Mutational Analysis of the Role of the Nudix
Motif in NUDT9 ADPR-pyrophosphatase Activity--
We have
previously reported an initial characterization of NUDT9 and the
isolated TRPM2 NUDT9-H domain as specific ADPRases (1). As a member of
the Nudix family, it would be expected that NUDT9 enzymatic activity is
dependent on the presence of metal ions and an intact Nudix
motif (15, 16). To experimentally verify the dependence of NUDT9
catalytic activity on metal ions, we produced NUDT9 and the TRPM2
NUDT9-H in bacteria and purified them to homogeneity (Fig.
5A, left panel).
Analysis of their respective activities under standard conditions
produced the Michaelis-Menten plots shown in Fig. 5B,
left panels (Km and
Vmax from these plots were previously reported
in Ref. 1), and metal and pH optima and metal specificities derived
from ion or pH manipulations performed at saturating [ADPR] (2 mM) are shown in the right and middle
panels. As can be seen, both enzymes have maximal activity at 16 mM Mg2+ and in similar pH ranges, with NUDT9
but not the TRPM2 NUDT9-H having the ability to allow Mn2+
to substitute for 50% of maximal activity at 5 mM. To
experimentally verify the role of the Nudix motif in NUDT9 catalytic
activity, we performed a mutational analysis of Nudix motif critical
residues (Fig. 5C). We first mutated the terminal EE
residues of the Nudix motif of NUDT9 to lysines, produced the protein
in E. coli, purified it as for wild type NUDT9, and assayed
its enzymatic activity. The EE
KK substitutions eliminated
detectable ADPRase activity, confirming the requirement for an intact
Nudix motif in NUDT9 catalytic function. We had previously observed
that although the TRPM2 NUDT9-H domain has ADPRase activity, it is two
orders of magnitude lower than that of NUDT9 (1). We had therefore
speculated that two Nudix motif substitutions found in the NUDT9-H of
TRPM2 (REF
RIL and EE
QE) accounted for its markedly lower
level of activity relative to NUDT9. To test the relevance of these substitutions, we produced NUDT9 versions with various combinations of
the Nudix motif substitutions found in the TRPM2 NUDT9H, purified them,
and assayed their activity at saturating [ADPR] (2 mM). The REF
RIL substitution greatly reduced detectable NUDT9
activity in our assay (to ~1% of wild type), whereas the EE
QE
substitution caused little or no detectable change in activity of
either wild type or RIL versions. We therefore conclude that the
REF
RIL substitution found in the TRPM2 NUDT9-H is sufficient to
account for the loss of activity of the NUDT9-H domain relative to
NUDT9. The REF
RIL and EE
KK mutant enzymes were as stable
as WT NUDT9 and easily purified, suggesting that they fold normally into a stable three-dimensional structure and that the effect of the
mutations is due to alterations within or nearby the NUDT9 active site.
This conclusion is further supported by results from partial
proteolysis of these enzymes (see below).

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Fig. 5.
Purification and analysis of NUDT9 activity.
A, purification of NUDT9 and NUDT9H. Shown are
Coomassie-stained gels of lysates of bacteria expressing NUDT9
(left panel) or the TRPM2 NUDT9-H (right panel)
before induction (non), after induction (I), and
after subsequent purification (P). B,
Michaelis-Menten plots, metal-ion and pH optima, and metal-ion
specificity of NUDT9 and NUDT9-H. C, mutational analysis of
NUDT9 activity. Shown in the table are the amino acid
sequences of the Nudix motif (left column) and the measured
enzymatic activity at saturating [ADPR] (right column) of
various NUDT9 constructs. The first construct is wild type NUDT9; the
structure of the TRPM2 Nudix motif is shown in the box just
below the table.
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Partial Proteolysis of WT NUDT9 and REF
RIL and EE
KK
Mutant Enzymes Reveals a Core Enzyme with ADPRase
Activity--
Because site-directed mutagenesis of enzymes can produce
altered protein folding, which hinders clear interpretation of the effects of a particular mutation on enzymatic activity, we compared partial proteolytic patterns of wild type, REF
RIL and
EE
KK enzymes. All three enzymes gave an identical pattern
consisting of the appearance of a major fragment (F1) of molecular mass
22 kDa (Fig. 6A,
left and middle panels), suggesting that the
mutant enzymes fold into the same basic conformation as WT NUDT9. We purified the F1 fragment for WT NUDT9, and obtained N-terminal and
internal protein sequences from it (Fig. 6B,
underlined sequences). These protein sequences identified
the fragment as extending from residue 164 of the NUDT9 coding sequence
to near the end of the predicted protein, including the Nudix motif
where our mutations were targeted. An expression vector was constructed
for the WT NUDT9 F1 fragment, and it was expressed and purified. Assays
of this preparation of purified F1 fragment for ADPRase activity demonstrated that it retained a high degree of specificity (its activity toward the close ADPR homologue ATP remained undetectable) and
had an identical Vmax as compared with the wild
type protein, although it had a significantly reduced
Km (see V (reaction velocity) versus S
(substrate concentration) plot, Fig. 6A,
right panel). That both WT and mutant enzymes produce
the same proteolysis-resistant fragment and that this fragment retains
specific ADPRase activity together provide strong support for the
conclusion that the RIL and KK mutations alter NUDT9 activity by virtue
of direct effects on the enzyme active site. The fact that the
"core" C-terminal fragment retains both activity and specificity
but has a substantially reduced Km further suggests
that NUDT9 is a multi-domain enzyme in which the N-terminal domain acts
to enhance the binding affinity of NUDT9 for ADP-ribose, whereas the
C-terminal domain provides catalytic specificity and activity.
The N-terminal domain may in addition play other specialized regulatory
functions. One such possibility is functioning as an interaction domain
for targeting NUDT9 ADPRase activity to a specific
microenvironment.

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Fig. 6.
Partial proteolysis of purified wild type and
mutant NUDT9 proteins. A, left panel, time
course of the partial proteolysis of purified WT NUDT9 with the
protease bromelain, using 15 µg NUDT9 and 30 ng bromelain per lane.
The obtained subfragments are designated F1 and F2. Middle
panel, time course of proteolysis of WT NUDT9 and point mutants
mutants KK and RIL/QE using the same conditions as for A. Right panel, V versus S plot for activity of wild
type NUDT9 (line with circles, averaged data from Fig. 2)
and purified recombinant F1 subfragment (triangles).
B, results of the mass-spectrometric analysis of the
obtained subfragments F1 and F2 shown under A by MALDI mass
spectrometry. Identified peptides after in-gel trypin digestion
of the F1 and F2 fragments are underlined in dark
gray (F1) or light gray (F2), demonstrating
that F2 is a degradation fragment of F1. The black
arrow shows the N terminus of the F1 fragment as determined by
Edman-degradation chemistry of the western-blotted F1 fragment.
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DISCUSSION |
The analyses of NUDT9 reported here indicate that there are highly
conserved NUDT9 homologs present in the most widely studied multicellular eukaryotic organisms, and that, in vertebrates at least,
NUDT9 predominantly functions in monomeric form within mitochondria
utilizing either Mg or Mn as a metal ion cofactor bound to the Nudix motif.
Several types of human ADPRases have been isolated based on procedures
for purification of ADPRase activities from cell and organellar
extracts. One of these, designated ADPRase-m, has been purified from
mitochondria and characterized in some detail (7). Several lines of
evidence suggest that this enzyme is likely to be the protein product
encoded by NUDT9
. First, mitochondria had ADPRase activity, which
eluted as a single peak during ion exchange and size exclusion
chromatography, suggesting that they contain only a single ADPRase
enzyme (7). Second, the purified protein had a predicted molecular mass
of 35 kDa (7), similar to the predicted molecular mass of 39,121 for
NUDT9
, particularly given that the post-translational proteolytic
processing observed for NUDT9
would reduce the size of the native
enzyme isolated from mitochondria. Although the Km
reported for this enzyme was 2-3 µM, far lower than the
100 µM Km we have observed for
recombinant NUDT9 (1, 7), this may be due to differences in assay
conditions or altered properties of the recombinant enzyme due to its
not having undergone the same post-translational proteolytic processing
as the endogenous enzyme. A 39-kDa cytosolic highly specific ADPRase
(ADPRase-I) was also reported by this same group (18), which is
significantly more than the 33 kDa size predicted for the
product of the NUDT9
transcript or for the processed forms of
NUDT9
. Determining whether ADPRase-I represents the product of the
NUDT9
transcript, alternative translational initiation from a
downstream methionine in full-length NUDT9
transcripts,
partially/differentially processed NUDT9
, or the product of a gene
other than NUDT9 will require further investigation.
Recent structural data from the E. coli ADPRase (17)
indicate that it functions as a dimer in which the interacting surface of one subunit provides part of the substrate recognition surface for
catalysis mediated by the active site of the other subunit. In
contrast, our analyses suggest that NUDT9 is active as a monomer. This
is consistent with the marked sequence divergence between NUDT9 and
other classic ADPRases exemplified by the E. coli, S. cerevesiae, and human NUDT5 sequences included in the phylogenetic analysis of Fig. 1. Based on the presence of highly conserved regions
on either side of the conserved Nudix catalytic site of NUDT9
(dark gray boxed areas in Fig. 1), it seems likely that interactions between ADPR and these regions of NUDT9 account for the
high degree of enzymatic specificity exhibited by NUDT9. The results of
the partial proteolysis, in which a core fragment from the
C-terminal region of NUDT9 including these regions was found to retain
substantial specific ADPRase activity also support this conclusion and
furthermore suggest that NUDT9 has a proteolytically labile N-terminal
domain, which enhances the affinity of the C-terminal domain for
ADPR.
Our mutational analysis confirms the role of the Nudix motif in the
ADPRase activity of NUDT9, and provides insight into the relationship
between NUDT9 and the NUDT9-H domain found in TRPM2. We had previously
speculated that the REF
RIL and EE
QE substitutions accounted for the decreased enzymatic activity of the NUDT9-H domain
relative to NUDT9 (1). Our present mutational analysis suggests that
the REF
RIL substitution is the most important determinant of the
decreased activity, and that the EE
QE substitution would not
substantially affect the activity of the TRPM2 NUDT9-H. As an
additional point, previous work (19, 20) on Nudix motif mutations in
the context of other NUDIX enzymes has suggested that mutation of the
glutamate residue in the REF motif would eliminate metal-ion binding
but otherwise leave the enzyme structure intact, an idea further
supported by the E. coli ADPRase structure, where the Nudix
motif is positioned for involvement in catalysis but not substrate
recognition. These observations add credence to the hypothesis that the
NUDT9-H of TRPM2 is an evolutionary adaptation of NUDT9 as an
interaction domain for ADPR-mediated gating of TRPM2.
The demonstration that human NUDT9 is to a large extent likely to be
compartmentalized specifically to mitochondria, the high level
conservation among vertebrate NUDT9 homologs of the signal sequence
most likely involved in mitochondrial targeting of NUDT9, and the
observation that NUDT9 homologs are found only in multicellular eukaryotes suggests that vertebrates and possibly other multicellular eukaryotes possess mitochondrial metabolic processes that result in the
production of free ADPR. Whether such processes are unique to
multicellular eukaryotic mitochondria remains to be determined, although the presence of the highly specific ADPRase in
M. jannaschii suggests that production of free
ADP-ribose is not a unique feature of eukaryotic metabolism. In either
case, the previous isolation of ADPRase activity from mitochondria, our
cloning and characterization of NUDT9 as a specific ADPRase, and
previous demonstrations of ADP-ribose production by isolated rat liver
mitochondria provide a strong argument that ADP-ribose production in
mitochondria is a physiologically important phenomenon in multicellular
eukaryotes (21, 22). At present the role (if any) of free ADP-ribose produced by mitochondria in intact cell physiology or pathophysiology is currently unclear. An intriguing possibility, raised by our present
results and our previous demonstration that the TRPM2 ion channel is
gated by ADPR, is that mitochondrial production of ADP-ribose serves as
a signaling mechanism for inducing the entry of extracellular Na and Ca
via gating of TRPM2.