Cloning, Expression, and Characterization of a Human Inosine Triphosphate Pyrophosphatase Encoded by the ITPA Gene*

Shengrong LinDagger §, Alexander G. McLennan||, Kang YingDagger , Zhao WangDagger , Shaohua GuDagger , Hua JinDagger §, Chaoqun WuDagger , Weiping LiuDagger , Youzhong YuanDagger , Rong TangDagger , Yi XieDagger **, and Yumin MaoDagger **

From the Dagger  State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, People's Republic of China, § United Gene Holdings, Ltd., Shanghai 200092, People's Republic of China, and the || Cell Regulation and Signalling Group, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom

Received for publication, December 11, 2000, and in revised form, February 22, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ITP and dITP exist in all cells. dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC 3.6.1.19). Here we report the cloning, expression, and characterization of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an enzyme whose activity has been reported in many animal tissues and studied in populations but whose protein sequence has not been determined before. At the optimal pH of 10.0, recombinant hITPase hydrolyzed ITP, dITP, and xanthosine 5'-triphosphate to their respective monophosphates whereas activity with other nucleoside triphosphates was low. Km values for ITP, dITP, and xanthosine 5'-triphosphate were 0.51, 0.31, and 0.57 mM, respectively, and kcat values were 580, 360, and 640 s-1, respectively. A divalent cation was absolutely required for activity. The gene encoding the hITPase cDNA sequence was localized by radiation hybrid mapping to chromosome 20p in the interval D20S113-D20S97, the same interval in which the ITPA inosine triphosphatase gene was previously localized. A BLAST search revealed the existence of many similar sequences in organisms ranging from bacteria to mammals. The function of this ubiquitous protein family is proposed to be the elimination of minor potentially mutagenic or clastogenic purine nucleoside triphosphates from the cell.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ITP has been found in many animal tissues. It is generated by pyrophosphorylation or stepwise phosphorylation of IMP, an essential metabolite of purine biosynthesis and a precursor of both AMP and GMP (1-3). The deoxyribonucleotide dITP may be generated from dATP by slow, non-enzymatic hydrolysis or by reduction of ITP (3). Normally, the cellular ITP/dITP concentration is very low. The inability to demonstrate the synthesis of ITP/dITP in cellular preparations has been attributed to the presence in the cytoplasm of an inosine triphosphatase (ITPase),1 an enzyme that does not permit accumulation of these nucleotides (2).

ITPase hydrolyzes ITP/dITP to IMP/dIMP and PPi. XTP is also a substrate, but activity toward other purine nucleoside triphosphates is low, whereas no activity is found with IDP or IMP (2-6). Different studies have measured different Km values for ITP (2, 6-12), but in all cases an alkaline pH optimum and an absolute requirement for a divalent ion such as Mg2+ or Mn2+ has been found. The range of ITPase activity in erythrocytes from Caucasian populations has been measured as 120-320 µmol of IMP produced/h/g Hb. In one population study, evidence was presented that a deficiency of ITPase was responsible for the high level of ITP found in 7 of >6,000 samples from mainly unrelated individuals. The frequency of heterozygosity for ITPase deficiency in Caucasian populations is estimated to be ~5% (6, 13-15). The gene coding for ITPase, ITPA, is located on the short arm of chromosome 20 (16).

Although mammalian ITPase activities have been identified in human erythrocytes (9), rabbit liver (10), and several rat tissues (17), no gene has been cloned and characterized. Recently a novel bacterial nucleoside triphosphate pyrophosphatase Mj0226, from Methanococcus jannaschii, was revealed by structure-based identification and subsequent biochemical analysis (18). In the presence of Mg2+ or Mn2+ ions Mj0226 can efficiently hydrolyze non-standard nucleotides such as XTP to xanthosine 5'-monophosphate and ITP to IMP but not the canonical standard nucleotides. A yeast protein, Ham1p, was reported to be the product of a gene controlling sensitivity of yeast strains to 6-N-hydroxylaminopurine (HAP) (19). This yeast protein is homologous to the Mj0226 protein, with about 30% sequence identity. Experiments with yeast HAM1- mutants have shown that adenine-requiring haploid strains are unable to grow on HAP as the sole adenine source (19, 20), whereas Escherichia coli transformed with the HAMI gene are resistant to HAP mutagenesis (21). This phenomenon presumably occurs because the conversion of HAP to HAP triphosphate and subsequent incorporation into DNA is lethal (20). Based on biochemical information and sequence similarity, these two proteins may be microbial ITP hydrolases that can convert mutagenic, non-canonical purine nucleoside triphosphates to monophosphates and thus protect DNA from the incorporation of modified purine bases.

Here we report the cloning, expression, and characterization of a human ITPase, hITPase, that converts ITP and dITP to their respective monophosphates and PPi. The RNA transcription profiles of hITPase were determined by Northern blot and cDNA microarray analysis. The chromosomal localization of the hITPase gene, ITPA, is also described.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- dITP was from Roche Molecular Biochemicals, and other dNTPs were from Promeaga. ITP, XTP, IDP, IMP, and other NTPs were from Sigma. Advantage cDNA polymerase mix was from CLONTECH, and all other enzymes were from New England Biolabs.

Identification and Cloning of hITPase cDNA-- A high quality cDNA library was constructed using human fetal brain poly(A)+ RNA and a SMART PCR cDNA library construction kit (CLONTECH). cDNA synthesis was performed from 100-200 ng of mRNA, followed by 20-24 cycles of PCR amplification. The cDNAs were inserted into the pBlueScript SK plasmid vector and transformed into E. coli DH5alpha by electroporation (Gene Pulser; Bio-Rad). A 96-well R.E.A.L. plasmid kit (Qiagen) was used to prepare double-stranded plasmid DNA. Sequencing reactions were performed with Big-Dye primer cycle sequencing and Big-Dye terminator cycle sequencing Kits (PerkinElmer Life Sciences) with the -21M13 or M13Rev primers to obtain the 5' or 3' sequences. The complete sequences were determined and confirmed by primer walking using the Big-Dye terminator cycle sequencing kit. Sequencing was performed on a PE-ABI 377 sequencer. The Acembly program was used to assemble the full-length cDNA sequences. Vector sequences were removed, and data base searches were performed with the BLAST using the NCBI web site.

Expression in E. coli and Purification of Human ITPase-- The hITPase cDNA was cloned between the BamHI and HindIII sites of the bacterial expression vector pQE31 (Qiagen) after PCR amplification. Transformants of E. coli M15/pREP4 with the resulting pQE31- hITPase construct were grown at 30 °C in 100 ml of LB medium with 100 µg/ml ampicillin and 25 µg/ml kanamycin. The plasmid pREP4 constitutively expresses the Lac repressor protein encoded by the lacI gene to reduce the basal level of expression (Qiagen). When the culture had grown to an A600 of 0.6, isopropyl beta -D-thiogalactopyranoside was added to a final concentration of 1 mM. After inducing the expression of the hITPase protein for 4 h at 30 °C, cells were harvested, washed, and resuspended in 20 ml of 20 mM sodium phosphate buffer, pH 7.4, containing 300 mM NaCl and 10 mM imidazole. The cell suspension was sonicated, and the lysate was cleared by centrifugation at 20,000 × g and 4 °C for 20 min. The supernatant was then poured into a Ni2+-nitrilotriacetic acid-agarose column, washed with phosphate buffer containing 60 mM imidazole, and eluted with buffer containing 500 mM imidazole. The purity of the eluted protein was checked by SDS-PAGE. Fractions containing the recombinant hITPase were dialyzed overnight against two changes of 1 liter of 50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 50% glycerol. Dialyzed samples were stored at -20 °C.

Enzyme Assay-- Substrates were screened, and kinetic parameters for substrate hydrolysis were determined by measuring the Pi released in a coupled assay by coincubation of substrate with hITPase and inorganic pyrophosphatase (22). The standard assay (800 µl) for triphosphate nucleotide substrates involved incubation of 1.25 mM substrate with 15 ng of hITPase and 1 unit of inorganic pyrophosphatase for 10 min at 37 °C in 50 mM Tris-HCl, pH 8.5, 50 mM MgCl2, 1 mM dithiothreitol. The Pi released was measured colorimetrically. Pi released from triphosphate nucleotide substrates in control assays without hITPase was subtracted.

Reaction products were identified by high performance liquid chromatography. Reaction mixtures described above but without inorganic pyrophosphatase were incubated at 37 °C for 10 min in a volume of 800 µl. The reaction was stopped by adding 200 µl of 20% trichloroacetic acid. Aliquots (10 µl) were injected onto a 4.6 × 250 mm Zorbax 300SB-C18 5-µm column (Hewlett Packard) at 30 °C. Chromatography conditions were as described previously (23).

Northern Blot Analysis-- Multiple tissue Northern (MTNTM) blots containing 2 µg of poly(A+) mRNA isolated from a variety of human tissues were purchased from CLONTECH. Blots were probed with a full-length human ITPase cDNA that had been radioactively labeled with [alpha -32P]dCTP by random priming using a random primer labeling kit (Amersham Pharmacia Biotech). Northern hybridization was performed according to the manufacturer's recommendations. The blot was hybridized at 68 °C overnight and washed in solution 1 (2× SSC and 1% SDS) three times at 65 °C and twice in solution 2 (0.1× SSC and 0.5% SDS) at 50 °C. The blot was stripped by incubation for 10 min in 0.5% SDS at 95 °C and reprobed with radiolabeled beta -actin cDNA as an indicator of mRNA loading.

Chromosome Mapping of the ITPA Gene-- To determine the chromosomal localization of the human ITPA gene, the Stanford G3 radiation hybrid panel (Research Genetics) consisting of 83 hybrid cell lines was screened by PCR. The PCR primer pair used for amplification was 5'-CTCTGAGAAACTCTGGCAAGTGGACG-3' (forward primer) and 5'-CACGCCCTCACTCCCACCAAGTAC-3' (reverse primer), derived from the 3'-untranslated region of the human ITPA gene. PCR products, 133 base pairs in length, were identified by electrophoresis on a high resolution agarose gel. The results were submitted to the Stanford Human Genome Center for statistical evaluation.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification and Cloning of the Human ITPA Gene-- The GenBankTM non-redundant and EST data bases were searched with individual sequences from our collection of full-length assembled cDNAs using the NCBI BLAST server to identify sequences of interest to our laboratories. One such sequence, represented in our collection by three clones, was found to encode a putative protein similar to the M. jannaschii Mj0226 and Saccharomyces cerevisiae Ham1p proteins (amino acid identities of 33 and 42% and E-values of 5 × 10-21 and 2 × 10-29, respectively). We then searched the UniGene data base and found a cluster of similar expressed sequence tags located at chromosome 20p. A search of the OMIM data base with the terms nucleotide and 20p revealed that the previously described, but unsequenced, inosine triphosphatase ITPA gene mapped to 20p by population analysis. This suggested that our protein may be the ITPA gene product. Based on the confirmatory evidence outlined below, we have named this putative protein human inosine triphosphatase, or hITPase.

The longest hITPase cDNA clone we obtained had 1085 base pairs containing an open reading frame of 585 nucleotides from the first translation initiation codon ATG to the termination codon TAA (nucleotides 105-689; GenBankTM accession number AF219116). The 3'-end of the sequence contains a poly(A) stretch, preceded by a putative polyadenylation signal AATAAA (nucleotides 1062-1067). The open reading frame encodes a 194-amino acid protein with a predicted molecular mass of 21,501 Da and pI of 5.50. There is an in-frame stop codon upstream of the open reading frame whereas the proposed initiating ATG is in the highly favorable sequence context ACCATGG, indicating that the predicted sequence probably represents the full-length protein.

Expression and Purification of hITPase-- After E. coli M15/pQE31-ITPase was induced with isopropyl beta -D-thiogalactopyranoside, the 6× His-tagged recombinant hITPase yielded a major band with an apparent molecular mass of 25 kDa on an SDS-PAGE gel corresponding to about 15-20% of the total applied protein (Fig. 1). Purification by binding to Ni2+-nitrilotriacetic acid-agarose resin and elution with buffer containing 500 mM imidazole resulted in a near homogeneous preparation (Fig. 1). To confirm that this was the expected expression product, an accurate molecular mass of 23,072 Da was determined by mass spectrometry (data not shown). This result is very close to the predicted mass of 23,069 Da for the recombinant hITPase, which includes the N-terminal vector-derived tag sequence MRGSHHHHHHTDP. The larger apparent molecular mass determined by SDS-PAGE may be because of incomplete heat denaturation and SDS binding.


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Fig. 1.   SDS-PAGE of E. coli extracts expressing hITPase and of the purified enzyme. E. coli M15 cells transformed with pQE31-hITPase were induced with 1 mM isopropyl beta -D-thiogalactopyranoside for up to 4 h. Lanes 1-5, aliquots were taken at hourly intervals from 0 to 4 h, analyzed on a 12% SDS gel, and stained with Coomassie Blue. Lane 6, hITPase protein after purification on a Ni2+-nitrilotriacetic acid-agarose column as described under "Experimental Procedures." The protein standards were rabbit phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), rabbit actin (43 kDa), bovine carbonic anhydrase (31 kDa), trypsin inhibitor (20 kDa), and hen egg white lysozyme (14.4 kDa).

Properties of the Enzyme-- Activity of the hITPase toward inosine nucleotides (ITP, IDP, IMP, and dITP), XTP, and the eight canonical ribo- and deoxyribonucleoside triphosphates was determined. At a fixed concentration of 1.25 mM, ITP, dITP, and XTP were the best substrates for the recombinant hITPase followed by GTP and dGTP (Fig. 2). In the absence of inorganic pyrophosphatase, no Pi production was detected by the colorimetric assay. The hITPase showed very low activity toward other nucleoside triphosphates; UTP, (d)CTP, TTP, and (d)ATP were hydrolyzed 10-100-fold less than dITP (Fig. 2). IDP and IMP were not hydrolyzed at all, showing that hITPase is a nucleoside triphosphate pyrophosphohydrolase. High pressure liquid chromatography analysis confirmed the production of IMP and dIMP as primary products; no IDP or dIDP was observed (data not shown).


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Fig. 2.   Substrate specificity of hITPase. Purified hITPase was incubated with 1.25 mM substrate at 37 °C for 10 min in 50 mM Tris-HCl, pH 8.5, 1 mM DTT, 50 mM MgCl2. The hydrolysis of the substrates was assayed using the colorimetric procedure described under "Experimental Procedures" and depicted relative to the activity with XTP. Each result is the mean ± S.D. from three experiments.

Similar to the purified human erythrocyte ITPase, recombinant hITPase had an absolute requirement for Mg2+ ions and a high Mg2+ optimum (6, 12). Activity increased sharply up to about 10 mM MgCl2 and reached a plateau between about 30 and 100 mM (data not shown). The recombinant hITPase also had a markedly high pH optimum of ~10.0 in glycine buffer, similar to the value of 9.6 from previous reports (12). Activity in the absence of 1 mM dithiothreitol was about half that in its presence indicating a requirement (though not absolute) for reducing conditions (data not shown). When analyzed by gel filtration, the recombinant 23-kDa tagged hITPase behaved as a homogeneous 45-kDa dimer (Fig. 3).


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Fig. 3.   Molecular weight determination of native recombinant hITPase by gel filtration chromatography. A sample of homogeneous hITPase protein was applied to a 10 600-mm Superdex 75 column (Amersham Pharmacia Biotech) in 0.05 M sodium phosphate buffer, pH 7.3, 0.1 M Na2SO4 and eluted at 0.5 ml/min in the same buffer. Fractions were assayed for enzyme activity as described under "Experimental Procedures." The column was calibrated with the following standards under the same conditions: A, bovine serum albumin, 68 kDa; B, hen ovalbumin, 45 kDa; C, bovine chymotrypsinogen A, 25 kDa; D, cytochrome c, 12.5 kDa. The elution position of the hITPase and the V0 are marked with arrows.

With ITP, dITP, and XTP as substrates, the recombinant hITPase followed Michaelis-Menten kinetics but with clear evidence of inhibition at higher substrate concentrations, most easily seen in reciprocal Lineweaver-Burk plots of the data (Fig. 4). Both substrate inhibition and inhibition by contaminating (deoxy)nucleoside diphosphates have been documented before for this enzyme (6, 12). For this reason, kinetic parameters were calculated by non-linear regression using data points only at concentrations that did not appear to result in inhibition according to visual analysis of the reciprocal plots (Table I). Within the errors of the kcat/Km values calculated in this way, all three substrates appear to be used with similar efficiencies.


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Fig. 4.   Lineweaver-Burk plots for the hydrolysis of ITP, dITP, and XTP by hITPase. Enzyme assays were performed with 15 ng of enzyme over the substrate range 0.056-3.35 mM, and the products were quantified by the colorimetric procedure described under "Experimental Procedures." Values are the means of four separate determinations. Error bars are omitted for clarity.

                              
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Table I
Kinetic parameters of recombinant human ITPase
Kinetic constants were determined from data obtained using the colorimetric assay described under "Experimental Procedures" using data points only at concentrations that did not appear to result in substrate or product inhibition according to visual analysis of reciprocal plots. Figures are mean ± S.D. (n = 4).

Tissue-specific Expression of hITPase-- To determine the size and distribution of hITPase mRNA transcripts, Northern blots containing 2 µg of poly(A)+ RNA from different adult human tissues were probed with [32P]dCTP-labeled hITPase cDNA. This revealed a transcript of about 1.4 kb in all 24 adult human tissues examined, with the most abundant expression in heart, liver, sex glands, thyroid, and adrenal gland (Fig. 5). These results were largely confirmed by cDNA microarray analysis, which showed heart, liver, thyroid, and thymus tissues to have the highest normalized expression ratios of those examined compared with housekeeping genes (all >2.6; data not shown).


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Fig. 5.   Expression of hITPase mRNA in human tissues. Northern blots of poly(A)+ RNA from 23 human tissues (2 µg per lane) were hybridized with 32P-labeled full-length hITPase cDNA probe. The membranes were stripped and reprobed with a beta -actin cDNA probe. Lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas; lane 9, spleen; lane 10, thymus; lane 11, prostate; lane 12, testis; lane 13, ovary; lane14, small intestine; lane 15, colon; lane 16, peripheral blood leukocyte; lane 17, stomach; lane 18, thyroid; lane 19, spinal cord; lane 20, lymph node; lane 21, trachea; lane 22, adrenal gland; lane 23, bone marrow.

Sequence Comparisons-- There are more than 40 protein and putative protein sequences in GenBankTM that have moderate similarity to human ITPase. All of these proteins are about 200 amino acids long and have similar secondary structures as predicted by the PHDsec program (24). Multiple sequence alignment of hITPase and related sequences revealed a new conserved protein family representing inosine triphosphatases from bacteria to mammals. In the alignment (Fig. 6), the amino acid residues corresponding to positions comprising the nucleotide binding site predicted by three-dimensional structural analysis of Mj0226 are shown (18). Not all are conserved; however, in some cases the side chain may be of lesser importance than the backbone amide in binding the substrate (18).


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Fig. 6.   Sequence alignment of hITPase with homologous proteins or putative proteins. Full-length hITPase and homologous protein or putative protein sequences were aligned using the Clustal W program and displayed using Genedoc software. Sequences are from (with GenBankTM accession numbers in brackets) Homo sapiens hITPase (AF219116), Mus musculus (W54379), Drosophila melanogaster (AE003608), Caenorhabditis elegans (U13642), Arabidopsis thaliana (T05241), S. cerevisiae Ham1p (S57088), M. jannaschii Mj0226 (C64328), Archaeoglobus fulgidus (E69529), Pyrococcus horikosii (C71206), E. coli (A65081), Bacillus subtilis (C69986), Hemophilus influenzae (D64146), and Deinococcus radiodurans (G75550). Black shading indicates 100% conservation of amino acid similarity, white on gray is 80-100%, black on gray is 60-80%, and no shading is <60% conservation. Amino acid residues comprising the predicted nucleotide binding site are marked with an asterisk.

Fig. 7 shows the dendrogram of the alignment results generated by the TREE-PUZZLE 5.0 program (25). For each internal branch, the bootstrap proportion was estimated by the Dayhoff method with 1000 replications (26). It is clear that these homologues fall into three divisions representing the three superkingdoms of eukaryota, archaea, and eubacteria and that the ITPase family, therefore, has an ancient origin and function.


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Fig. 7.   Phylogenetic tree of hITPase and homologous sequences from other organisms. The TREE-PUZZLE program with the Dayhoff model was applied. The length of each branch is proportional to the estimated number of amino acid substitutions. The numbers at the internodes represent the bootstrap values of each branch.

Chromosomal Localization-- The chromosomal location of the human ITPA gene was determined with the Stanford G3 radiation hybrid panel with a 3'-untranslated region-specific primer pair for human ITPA gene. The expected 133-base pair PCR product was amplified from 12 of 83 DNAs from the hybrid cell lines. Data were submitted to the Stanford Human Genome Center for statistical evaluation. The human ITPA gene was located on chromosome 20, close to markers SHGC-14786, SHGC-2800, and SHGC-21112 from the Stanford G3 chromosome 20 radiation hybrid map data and between microsatellite marker AFM240zf4 (D20S181) and microsatellite anchor marker AFM036ya3 (D20S97). Our cDNA clone corresponds to NCBI Unigene Hs.6817, which was located on the same interval D20S113-D20S97 on GeneMap '99. We found two genes, PTPRA (protein tyrosine phosphatase, receptor type, alpha polypeptide) and CENPB (centromere protein B), upstream and downstream, respectively, of Unigene Hs.6817 on the same region. These genes also flank the mouse Itpa gene. This result confirmed our conclusion that the cDNA clone encodes the human ITPA gene product, hITPase (Table II).

                              
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Table II
Human ITPA and mouse Itp chromosome linkage maps
Genetic and physical map locations of ITPA, Itp, and surrounding genes are shown. Data are from the NCBI Human-Mouse Homology Map. The Human Physical map location cR3000 is from GeneMap99. STS (sequence-tagged site) markers of human ITPA are from human Unigene Hs.6817.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report, we have described the isolation and characterization of a cDNA clone encoding a human ITP/dITP/XTP pyrophosphatase, hITPase, from a human fetal brain cDNA library. Initially, the library was searched for potential homologues of the M. jannaschii nucleoside triphosphate pyrophosphatase Mj0026. The cDNA thus obtained encodes a 195-amino acid protein with a predicted molecular mass of 21,501 Da and 33% amino acid identity to Mj0026. The chromosomal localization of the cDNA clone matches that of the previously described ITPA locus on the short arm of chromosome 20 between the PTPRA and CENPB genes. These results confirm our view that this clone encodes the human ITPA gene product. The presence of hITPase mRNA in all human tissues examined and its high expression in endocrine glands was shown by Northern blot analysis and by cDNA microarray hybridization. The distribution of ITPA transcripts in human tissues agrees with the distribution of ITPase enzyme activity in different rat tissues (17). These and other studies also suggested that the enzyme was located in the cytoplasm (6, 17). We have confirmed this location by transfection of COS-7 cells with a construct in which hITPase was fused to the C terminus of green fluorescent protein. A diffuse but clearly non-nuclear distribution of fluorescence was observed.2

Enzymological analysis of the recombinant human protein expressed from this cDNA clone showed a high specificity for the hydrolysis of ITP, dITP, and XTP to their respective monophosphates. Characteristics of the recombinant enzyme were similar to those from earlier reports of ITPase isolated from human tissues, except the Km value for ITP, which at 0.51 mM was higher than those previously reported, 0.13 (12) and 0.07 mM (6). Possible explanations for this discrepancy are (i) different levels of inhibitory IDP in the substrate preparations used, (ii) lack of a post-translational modification in the recombinant enzyme that is present in the enzyme isolated from tissues, or (iii) reduced affinity for ITP and other substrates caused by the N-terminal tag.

Three different human populations have been reported with respect to their ITPase activity. The first has high activity, the second has a mean activity exactly 25% that of the first, whereas the third population has very low activity. A theoretical explanation of this study is that normal (active) and mutant (inactive) alleles exist in the population and that the ITPase is only active as a dimer with two normal subunits (14, 15). In heterozygotes, only one of the four hypothetical dimers would be composed of two normal monomers, leading to 25% activity. Consistent with this hypothesis is our finding that recombinant hITPase behaves as a homogeneous dimer under physiological conditions. This agrees with results generated by structural analysis of Mj0226 (18). In ITPase-deficient populations, there is an obvious elevation of ITP concentration in erythrocytes and other cells, showing that hITPase may be the major enzyme responsible for regulating the ITP concentration in human cells. Although no clinical abnormalities have been reported to be associated with complete hITPase deficiency, one study has reported a significant reduction in tissue ITPase activity in paranoid schizophrenics (56.3 ± 5.5 µmol/h/g of Hb) compared with normal controls (94.1 ± 5.3 µmol/h/g of Hb) (p < 0.0002), and it has been suggested that the resulting elevated ITP may inhibit the activity of glutamate decarboxylase, the enzyme responsible for generating the neurotransmitter gamma -aminobutyric acid (27, 28).

ITP and dITP can be incorporated into RNA and DNA, respectively, by polymerases (29-32). As an unusual nucleoside in RNA, inosine arising from incorporation from ITP could lead to the same effects on RNA sequence-specific interactions as the inosine arising from RNA editing by ADARs, adenosine deaminases that act on RNA (33, 34). Therefore, effects on structure, translatability, and degradation rate are all possible. Further, because adenosine deamination can alter RNA structure, sequence-independent processes also could be affected.

The deoxyribonucleotide dITP behaves as a dGTP analogue and is incorporated opposite cytosine with about 50% efficiency. Both isolated nuclei and purified DNA polymerases rapidly incorporated dITP into DNA. In the presence of ATP, dITP is stabilized in extracts of nuclei (35) and E. coli (36), allowing the possibility that a small amount of dIMP will also be incorporated into DNA in vivo. Although hypoxanthine DNA glycosylase can remove the base from DNA (37), evidence has been presented that this enzyme only efficiently removes a hypoxanthine base from an I-T base pair, whereas removal from an I-C base pair is 15-20 times slower (38). Because of the relative stability of an I-C base pair, inosine can remain until the next round of DNA replication, increasing the risk of direct mutagenesis. In vitro polymerase studies have shown that the presence of dITP in reaction mixtures may induce a high frequency of mutation (39, 40). Other reports shows that ITP and IDP added to cell cultures can cause elevated rates of chromosomal structural aberrations (41, 42).

In human erythrocytes ITP is continuously synthesized and broken down at a relatively high rate, forming a futile cycle that has been proposed to regulate the concentration of ATP. Additionally, ITP appears to be a substrate for the cartilage pyrophosphorylase associated with articular calcium crystal deposition (43) and a substrate for receptor/G proteins to activate effector systems (44, 45).

Other naturally occurring unusual purine nucleoside triphosphates could be considered as possible substrates of hITPase, such as the triphosphates of HAP (46) and 2-amino-N6-hydroxyladenine (47). The HAM1 protein, the hITPase homologue in yeast, can control the sensitivity of yeast strains to HAP added in medium. Overexpression of HAM1 can protect E. coli from both the toxic and mutagenic effects of HAP (21). Therefore, Ham1p must be acting as a HAP triphosphate pyrophosphatase to prevent the incorporation of HAP into DNA (20). Given the possibility that HAP could be generated inside living cells (48) and induce mutations and chromosomal aberrations (49), a role for the ITPA family in the prevention of HAP-induced mutagenesis must be considered.

A BLAST search of protein data bases revealed hITPase/Mj0026/Ham1p homologues in most of the bacterial and eukaryotic organisms that have been fully or partially sequenced. Sequence alignment shows that the previously identified nucleotide binding sites are conserved in these proteins. The results of a phylogenetic analysis showed that these homologues fall into three divisions, in accordance with the three superkingdoms of eukaryota, archaea, and eubacteria. The widespread presence of hITPase homologues suggests an ancient origin for ITPases, perhaps arising after the appearance of de novo purine synthesis.

An interesting comparison may be drawn between hITPase and its homologues and the nudix hydrolase gene family. This family is composed of mostly small, soluble nucleotide phosphohydrolases that possess a sequence signature motif called the nudix box, formerly called the MutT motif, named after the antimutagenic E. coli mutT gene product (50-52). The MutT protein can hydrolyze 8-oxo-dGTP, a highly mutagenic oxidized nucleotide, to 8-oxo-dGMP and PPi (50, 51). Because of gene duplication, any one genome may have several nudix family members. There are 13 paralogs in E. coli, 5 in S. cerevisiae, and at least 15 in man, several of which have been characterized as nucleotide hydrolases with widely varying substrate specificity. Almost all of the substrates of nudix hydrolases possess a nucleoside diphosphate linked to another moiety, "x" (51). These enzymes are believed to eliminate toxic nucleotide derivatives from the cell and regulate the levels of important signaling nucleotides and their metabolites (51, 52). Unlike nudix family members, the ITPA family has only one member per genome without paralogs. This may indicate a simpler but still important role in cleansing minor nucleotides from the cell. Another example of a non-nudix nucleoside pyrophosphohydrolase that restricts the accumulation of a minor NTP is deoxyuridine 5'-triphosphatase, which both prevents the incorporation of uracil into DNA and generates dUMP for dTMP synthesis (53, 54).

Although the biochemical properties of ITPases have been well characterized, no definite biological function has yet been defined. One direction for future research is to study the toxicity and mutagenicity of its substrates ITP and dITP, as well as the other purine derivatives that may be hydrolyzed by ITPase in vivo. Confirmation of its cellular and disease-related function must await the result of gene knock-out studies and further population investigations.

    ACKNOWLEDGEMENT

We thank Yueqiong Chao from United Gene Holdings, Ltd. for providing facilities for DNA sequencing and Junxia Zhu and Xiongying Fang for technical assistance. Thanks are also because of Prof. Yang Zhong for help with calculating and constructing the dendrogram alignment and to Prof. Wanxiang Xu for helpful discussions.

    FOOTNOTES

* This work was supported by funds from United Gene Holdings, Ltd.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF219116.

Contributed equally to this paper.

** To whom correspondence may be addressed: Inst. of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China. Tel.: 86-21-65643573; Fax: 86-21-65642502; E-mail: yxie@fudan.edu.cn.

Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M011084200

2 S. Lin and J. Shi, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: ITPase, inosine triphosphate pyrophosphatase; HAP, 6-N-hydroxylaminopurine; XTP, xanthosine 5'-triphosphate; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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