| Nucleic Acids Research | Pages |
Multi-forms of human MTH1 polypeptides produced by alternative translation initiation and single nucleotide polymorphism
Introduction
Materials And Methods
Chemicals
Cells and culture
Antibodies
Site-directed mutagenesis
In vitro transcription and translation
Immunoprecipitation
Western blotting
Reverse transcription-polymerase chain reaction (RT-PCR)
Genomic PCR and direct sequencing
Screening of genomic alterations in exon 4 of the human MTH1 gene
Results
Multi-forms of MTH1 polypeptides in human cells
Multiple MTH1 polypeptides produced by in vitro translation of MTH1 mRNAs carrying exon 2b-2c segments
All of the four AUGs in B-type MTH1 transcripts alternatively function as translation initiation sites in vitro
Polymorphic alteration in exon 2c of MTH1 gene determines the number of MTH1 polypeptides produced in vivo
Linkage disequilibrium between the two polymorphisms of MTH1 gene
Discussion
Multiple translation products of MTH1 mRNAs by alternative translation initiation
Functions of multi-forms of MTH1 proteins
Subcellular localization of MTH1 proteins
Genetic polymorphisms in MTH1 gene and their effects
Acknowledgements
References
Multi-forms of human MTH1 polypeptides produced by alternative translation initiation and single nucleotide polymorphism
Received August 23, 1999; Revised and Accepted October 4, 1999
ABSTRACT The human MTH1 gene for 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase, produces seven types (types 1, 2A, 2B, 3A, 3B, 4A and 4B) of mRNAs. The B-type mRNAs with exon 2b-2c segments have three additional in-frame AUGs in their 5[prime] regions. We report here that these transcripts produce three forms of MTH1 polypeptides (p22, p21 and p18) in in vitro translation reactions. Three polypeptides were also detected in extracts of human cells, using western blotting. B-type mRNAs with a polymorphic alteration (GU->GC) at the beginning of exon 2c that converts an in-frame UGA to CGA yielding another in-frame AUG further upstream, produced an additional polypeptide (p26) in vitro. Substitution of each AUG abolished the production of each corresponding polypeptide. Cell lines from individuals with the GC allele contain more B-type mRNAs than do those of GT homozygotes, and the former produce all of four polypeptides but the latter lack p26. Amounts of each polypeptide reflected copy number of the GC allele in each cell line. There is an apparent linkage dis-equilibrium between the two polymorphic sites, GT/GC at exon 2c and Val83/Met83 at codon 83 for p18.
INTRODUCTION
Oxygen radicals are generated during normal cellular metabolism and attack biologically important macromolecules, such as nucleic acids, proteins and lipids. Among many classes of DNA damage caused by oxygen radicals, an oxidized form of guanine base, 8-oxo-7,8-dihydroguanine (8-oxoguanine) appears to be most pertinent to mutagenesis and carcinogenesis (1-4). 8-Oxoguanine can pair with adenine as well as cytosine with an almost equal efficiency during DNA replication, and thus has the potential to cause a high frequency of mutation (5,6).
To minimize replication errors caused by 8-oxoguanine in DNA, living organisms have evolved efficient mechanisms for avoiding such errors. Escherichia coli has two types of DNA repair gene to avoid 8-oxoguanine-related error during DNA replication. The mutM gene encodes DNA glycosylase/lyase for 8-oxoguanine paired with cytosine (7-10), and the mutY gene encodes an adenine-DNA glycosylase which excises adenine paired with 8-oxoguanine (11-13). Mutants lacking MutM or MutY protein showed a 10-100-fold higher frequency of spontaneous occurrence of G:C->T:A transversion, compared to the level in the wild-type (7,11), indicating that these repair proteins are essential for avoiding such replication errors. MutM orthologs have been identified in various bacteria and in a higher plant, Arabidopsis thaliana, but not in yeast or animals (14).
In Saccharomyces cerevisiae, the OGG1 gene encodes 8-oxo-guanine DNA glycosylase/lyase (15), and increased mutation rates were observed in the OGG1-deficient strain (16), indicating that DNA damage such as 8-oxoguanine recognized by OGG1 is also mutagenic in eukaryotes. Enzymes similar to OGG1 and MutY proteins had been found in human cells (17,18), and recently several groups have cloned cDNAs for human mRNA which encodes a homologous protein to the yeast OGG1 protein (19-25). Although the human gene that encodes a protein homologous to E.coli MutY protein was also identified and designated as MYH (mutY homologue), biochemical function of the product has remained unknown (26).
Oxidation of guanine also proceeds in the form of free nucleotides, and an oxidized form of dGTP, 8-oxo-7,8-dihydro-2[prime]-deoxyguanosine 5[prime]-triphosphate (8-oxo-dGTP), is a potent mutagenic substrate for DNA synthesis (27). The MutT protein of E.coli hydrolyzes 8-oxo-dGTP to the corresponding nucleoside monophosphate, and lack of the mutT gene increases the occurrence of A:T->C:G transversion 1000-fold over the wild-type level (27-30). Moreover, it has been shown that E.coli RNA polymerase misincorporates 8-oxo-GTP into mRNAs yielding mutant forms of proteins recognized as non-genomic mutations in mutT-deficient cells. MutT protein efficiently hydrolyses 8-oxo-GTP, and thus minimizes errors during transcription as well as replication, caused by misincorporation of oxidized guanine nucleotides (31).
Among the mammalian 8-oxoguanine related error avoidance systems, the human MTH1 gene was identified first and has been studied most extensively (32-34). Expression of MTH1 cDNA in mutT- E.coli cells suppresses the elevated spontaneous mutation rate to an almost normal level, indicating that human MTH1 protein might have the same anti-mutagenic capacity as does the E.coli MutT protein (33,35). MTH1 mRNA is abundant in the human thymus, testis and the embryonic tissues.In peripheral blood lymphocytes, expression of MTH1 mRNA is induced after proliferative activation, suggesting that MTH1 expression is up-regulated in proliferative tissues (36). In human cells, MTH1 protein is localized mostly in the cytoplasm and with some in mitochondria, suggesting that MTH1 protein is involved in the sanitization of nucleotide pools both for nuclear and mitochondrial genomes (37).
The human MTH1 gene is located on chromosome 7p22, and consists of five major exons (35,36). We have shown that seven types of MTH1 mRNAs (types 1, 2A, 2B, 3A, 3B, 4A and 4B) with different 5[prime] sequences are produced by transcription initiation at different sites and by alternative splicing. In addition, all types of MTH1 mRNAs carry the entire coding region and three additional in-frame AUGs were found in the 5[prime] regions of B-type MTH1 mRNAs (36).
In the present work, we examined translation products from each MTH1 mRNA, and found that three forms of MTH1 polypeptides (p22, p21 and p18) are produced by alternative initiation of translation from B-type MTH1 mRNAs, both in vitro and in vivo. We also found a single nucleotide polymorphism in exon 2 of the MTH1 gene, an event that removes a termination codon in front of the initiation codon for p22 in B-type MTH1 mRNAs yielding the fourth MTH1 polypeptide, p26, translated from the most upstream AUG.
MATERIALS AND METHODS
Chemicals
[35S]methionine, 125I-labeled protein A and 14C-labeled methylated protein mixture were purchased from Amersham International PLC (Buckinghamshire, UK). Recombinant Taq DNA polymerase was obtained from Takara Shuzo (Kyoto, Japan). Other chemicals were obtained from Wako Pure Chemical Industries Ltd (Osaka, Japan).
Cells and culture
Lymphoblastoid cell lines established from healthy volunteers were kindly provided by Drs T. Tana and T. Sasazuki. Cell lines and peripheral blood lymphocytes were cultured as described previously (36).
Antibodies
Affinity-purified antibodies against recombinant human MTH1 protein (anti-MTH1) and a peptide corresponding to Lys38 to Val61 of human MTH1 protein (anti-M78) were prepared as described (37).
Site-directed mutagenesis
Each upstream ATG codon (ATG1-3) in type 2B (GC) MTH1 cDNA was replaced with an ATC codon by using QuikChangeTM site-directed mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). These cDNAs were designated as 1m, 2m and 3m type 2B (GC) MTH1 cDNA, respectively.
In vitro transcription and translation
Each type of MTH1 cDNA was placed under the control of the T7 promoter in pT7Blue vector (Novagen, Madison, WI). Plasmid pHYVal83-MTH1, in which the coding sequence from ATG4 (NcoI site in exon 3) to the termination codon with a polymorphism Val83 was also placed under the control of T7 promoter in pET8c expression vector, was as described (38). Transcripts were synthesized from BamHI linearized plasmids by T7 RNA polymerase at 30°C for 15 min using Single Tube Protein System 2 (Novagen). These mRNAs were translated in rabbit reticulocyte lysates in the presence of 50 µCi of [35S]methionine in a total volume of 50 µl at 30°C for 60 min.
Immunoprecipitation
Twenty microliters of in vitro translation products were diluted with 230 µl of RIPA buffer [150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 50 mM Tris-HCl (pH 8.0), 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml each of leupeptin, pepstatin and chymostatin]. Immunoprecipitation with anti-MTH1 was done as described (39), and protein A-Sepharose CL4B (Pharmacia Biotech, Uppsala, Sweden) was used to collect the immune complex. Precipitated antigen was separated by SDS-PAGE and radioactivity was measured in a Bio-Image analyzer BAS2000 (Fuji Photo Film, Tokyo, Japan).
Western blotting
Whole-cell extracts or tissue extracts were prepared in SDS sample buffer [0.125 M Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 4% 2-mercaptoethanol], subjected to SDS-PAGE then electro-transferred onto a nitrocellulose filter. Filters were blocked in 5% bovine serum albumin, 0.05% Tween-20, 10 mM Tris-HCl (pH 7.6), 150 mM NaCl at 52°C for 1 h and incubated with anti-MTH1 (2 µg/ml) in the blocking solution at 4°C for 12 h. The filters were incubated with 1 µCi/ml of 125I-labeled protein A and were washed, as described (39). Radioactivity was measured in a Bio-Image analyzer. Purified MTH1 protein (38) was used as a standard for quantification of MTH1 protein in extracts.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA (1 µg) was reverse-transcribed into single-stranded cDNA with MTH-P (5[prime]-CGGCCCCGAAGCCTCGCTTTTT-CAT-3[prime]) as a specific primer, using a First-Strand cDNA Synthesis kit (Pharmacia Biotech). To amplify a specific 5[prime] region for each type of MTH1 mRNA, PCR was done in 50 µl of reaction mixture containing the first-strand cDNA, 2.5 U of rTaq DNA polymerase, 10 µM sense primer for each type of MTH1 mRNA (T1-T4), 10 µM antisense primer MTH2-17 and 200 µM dNTP. The primers used are as follows: MTH2-17, 5[prime]-CCAGCACCAGGGTATAG-3[prime]; T1, 5[prime]-AGCGG-CGGTGCAGAACC-3[prime]; T2, 5[prime]-AAGCGGCGGTGCAGGTTT-3[prime]; T3, 5[prime]-AAGCGCGCGCGGGGATT-3[prime]; T4, 5[prime]-GGG-CTTTCTGTATCCCTAG-3[prime]. Initial denaturation was done at 94°C for 1 min, then amplification was performed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 2 min, followed by extension at 72°C for 2 min. The PCR products were analyzed by 8% PAGE.
Genomic PCR and direct sequencing
Genomic DNA was extracted from various types of human cells using ISOGEN kits (Nippon Gene, Toyama, Japan). To amplify the entire region for the MTH1 exon 2, PCR was done in 100 µl of reaction mixture containing 100 ng of genomic DNA, 10 µM of 668-5 (5[prime]-CCCTGCCTTATCGCAAGG-3[prime]) and 491-3 (5[prime]-GGCCATCAACTGATGGAAC-3[prime]), 200 µM dNTP and 2.5 U of rTaq DNA polymerase. The initial denaturation was performed at 95°C for 1 min, then amplification was done by 35 cycles of denaturation at 95°C for 45 s, annealing at 65°C for 45 s, and extension at 72°C for 45 s, followed by extension at 72°C for 3 min. The PCR product (254 bp) was purified using Microcon 100 (Amicon Inc., Beverly, MA) and subjected to direct sequencing using Dye Terminator Cycle Sequencing FS Ready Reaction kits and oligonucleotide GT-GC2 (5[prime]-GGCGGTCAGAGGAGAGC-3[prime]) as a primer. Nucleotide sequence was determined in an ABI373A DNA sequencer (Perkin-Elmer, Norwalk, CT).
Screening of genomic alterations in exon 4 of the human MTH1 gene
Genomic DNA was extracted from peripheral blood lymphocytes and cancerous or corresponding normal tissues from dissected specimens. Oligonucleotide primers labeled with 6-carboxyfluorescein hexachloride (HEX-452: 5[prime]-CAT-GGCACCATGCCCTGA-3[prime] for the intron 3 of human MTH1) and 6-carboxyfluorescein (FAM-432: 5[prime]-GAGATGGGACCC-GCATAG-3[prime] for the intron 4) were obtained from Kikotec Co. (Suita, Japan), and were used to amplify the 245 bp region containing the entire exon 4 of the human MTH1 gene. Genomic DNA (100-200 ng) was added into Multi-Ultra tube (Sorenson, BioScience Inc., Salt Lake City, UT) with final 50 µl of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 0.2 mM dNTPs, 0.2 µM each of primers and 1.25 U of rTaq DNA polymerase. The initial denaturation was done at 95°C for 1 min. Amplification was performed by 40 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 20 s and extension at 72°C for 30 s, followed by extension at 72°C for 1 min. Amplified products were subjected to 3% NuSieve 3:1 agarose (FMC BioProducts, Rockland, ME) gel electrophoresis and amounts of DNA fragments were estimated after staining by ethidium bromide. Appropriate amounts of the amplified product and a size marker, Genescan 2500 Rox (Perkin-Elmer) were prepared in 87% formamide, 5 mM EDTA, 5 mg/ml bluedextran (Mr 2 000 000; Sigma, St Louis, MO) and denatured by heating at 80°C for 7 min, then kept on ice. For single-strand conformation polymorphism (SSCP) analysis, an ABI373A DNA sequencer equipped with Genescan 672 software (Perkin-Elmer) was used to control electrophoresis and to collect data. Five microliters of denatured PCR product were loaded onto 5% Long Ranger gel (AT Biochem, Malvern, PA) made in 1× TBE [100 mM Tris-HCl (pH 8.1), 83 mM boric acid, 10 mM EDTA] with 5% glycerol, and run in 1× TBE at 1000 V for 8 h while keeping the temperature of the gel at 35°C using an external temperature control device (Astec, Fukuoka, Japan). Two types of SSCP for exon 4 corresponding to a base substitution from G (Val83) to A (Met83) at codon 83 of MTH1 (p18) were detected. Since the base substitution generates a new restriction site for NsiI, the existence of the Met83 allele was confirmed by digesting PCR fragments with NsiI.
RESULTS
Multi-forms of MTH1 polypeptides in human cells
We performed western blotting analysis of whole-cell extracts prepared from Jurkat and HeLa cells using anti-MTH1 antibodies combined with 125I-labeled protein A (Fig. 1A). In both cell lines, three immunoreactive bands corresponding to 21.8 (p22), 20.7 (p21) and 18 kDa (p18) polypeptides, were detected. p18 was the most abundant among the three MTH1 polypeptides, in agreement with the fact that the purified human 8-oxo-dGTPase is an 18 kDa polypeptide (32,33). To confirm that the three polypeptides are indeed products of the MTH1 gene, we performed immunoprecipitation of the MTH1 polypeptides from [35S]methionine-labeled Jurkat cells using the anti-M78, which recognizes the conserved residues (Lys38 to Val61) among MTH1 homologs (37), as well as the anti-MTH1. Essentially the same results were obtained with both antibodies, as seen in western blotting (our unpublished data).
Figure 1. Multiple MTH1 polypeptides exist in vivo. (A) Western blotting of human cell lines. Whole-cell extracts were subjected to SDS-PAGE. Western blotting was performed using anti-MTH1 antibodies. Bound antibodies were detected with 125I-labeled protein A. Radioactivity was measured in a Bio-Image analyzer. Lane 1, Jurkat 5 × 105 cells; lane 2, HeLa S3 5 × 105 cells; lanes 3-8, various amounts of bacterially expressed human 18 kDa MTH1 protein. (B) Standard curve for MTH1 protein quantitation. Correlation between the amounts of purified 18 kDa MTH1 protein and radioactivities measured is shown. PSL, photo-stimulated luminescence. Results from two independent experiments are plotted. Arrows indicate radioactivity for 18 kDa MTH1 protein of Jurkat (J) and HeLa S3 (H).
To estimate the amount of each MTH1 polypeptide, purified recombinant MTH1 (p18) was used as the standard in quantitative western blotting (Fig. 1A, lanes 3-8). A quantitative measurement of the protein was made by scanning the band using an image analyzer. As shown in Figure 1B, there is a good linear relationship in a range from 1 to 10 ng of recombinant p18. Thus, we estimated that a single cell of Jurkat or HeLa contains 4 × 105 or 2 × 105 molecules, respectively, of the p18. Radioactivities of bands for p22 and p21 were ~10% of those for p18 in each cell line, indicating that each single cell contains more than 1 × 104 molecules of p22 and p21.
Multiple MTH1 polypeptides produced by in vitro translation of MTH1 mRNAs carrying exon 2b-2c segments
As shown in Figure 2, seven types of MTH1 mRNAs with different 5[prime] sequences carry the entire coding region initiated at the AUG4, and the B-type mRNAs (2B, 3B and 4B) possess three additional upstream AUGs (uAUGs: AUG1, AUG2 and AUG3) in their 5[prime] regions, all of which are in the same reading frame as that of the coding region from the AUG4. Since the AUG1 is followed by a termination codon between AUG1 and AUG2, the two initiation codons (AUG2 and AUG3) as well as the AUG4 may be functional. Based on the cDNA sequences, we expected that A-type MTH1 mRNAs encode a polypeptide with a molecular mass of 17 939 Da, and that B-type MTH1 mRNAs may encode polypeptides with a molecular mass of 20 282 and 19 454 Da, in addition to the former.
Figure 2. Schematic representation of MTH1 mRNAs and predicted translational products from each AUG codon. (A) Genomic structure and alternative splicing of the MTH1 gene. In the upper part, the overall structure of the human MTH1 gene located on chromosome 7p22 is shown. Each colored box represents an exon. In the lower part, seven types of MTH1 mRNA, which are produced by alternative transcription initiation and splicing, are shown together with a part of the genomic structure. Alternative splicing is not observed in exons 3-5. Polymorphic alterations (GT->GC located at the beginning of exon 2c segment; GTG83->ATG83 in exon 4) are shown. (B) Types 1, 2A, 3A and 4A MTH1 mRNAs and their translational products. (C) B-type MTH1 mRNAs with the GU polymorphism at the beginning of exon 2c and their translational products. (D) B-type MTH1 mRNAs with the GC polymorphism at the beginning of exon 2c and their translational products. The lines show MTH1 mRNAs. Each colored box on the lines represents an exon shown in (A). AUG codons in-frame to the 18 kDa MTH1 protein coding region are shown above the colored boxes. The UGA codon in-frame to AUG1 at the polymorphic site is underlined. Boxes below the lines represent translational products. Closed boxes show translational products in-frame to 18 kDa MTH1 protein. The predicted molecular weights of translation products are shown at the right end of the line. The hatched box in (C) shows an upstream open reading frame.
To examine the possibility that these uAUGs serve as initiation codons for translation, each type of MTH1 mRNAs prepared in vitro using T7 RNA polymerase, was subjected to an in vitro translation reaction in rabbit reticulocyte lysate. Translated polypeptides were labeled with [35S]methionine and analyzed by SDS-PAGE after immunoprecipitation with anti-MTH1 (Fig. 3). When type 1 and A-type (2A, 3A and 4A) MTH1 transcripts were translated, only an 18 kDa polypeptide, which is likely to correspond to the p18 detected in Jurkat and HeLa cells, was detected (Fig. 3, lanes 3-5 and 8), as was the mRNA carrying only the coding region from the AUG4 translated (Fig. 3, lane 2). In vitro translation of B-type (3B and 4B) mRNAs produced two more MTH1 polypeptides, with apparent molecular masses of 21.8 and 20.7 kDa and which are almost identical to the p22 and p21 detected in Jurkat and HeLa cells (Fig. 1), in addition to the p18 (Fig. 3, lanes 6 and 7).
Figure 3. In vitro transcription/translation products of human MTH1 cDNAs. T7 transcripts of wild-type MTH1 cDNAs were translated in a reticulocyte cell-free system in the presence of [35S]methionine. Products were followed by immunoprecipitation with purified anti-MTH1 antibody and analysis by 15% SDS-PAGE. M, molecular weight marker. Lane 1, no template cDNA; lane 2, pET8c-MTH1 cDNA; lanes 3-8, type of MTH1 cDNA is shown above the lane.
All of the four AUGs in B-type MTH1 transcripts alternatively function as translation initiation sites in vitro
We reported a single nucleotide polymorphism at the 5[prime] splicing site (GT->GC) of the exon 2c segment, which alters the splicing pattern of exon 2c (36). As shown in Figure 2C, this polymorphic site is located between AUG1 and AUG2, and the AUG1 in B-type MTH1 mRNAs with the GU sequence is followed by a termination codon (GUGA) at this position, which is not the case for those with the GC sequence; this converts the termination codon to CGA and generates an extended open reading frame for a polypeptide of molecular mass 22 505 Da. To examine the possibility that the AUG1 is also functional as an initiation codon, type 2B (GC) MTH1 mRNA was subjected to in vitro translation. As shown in Figure 4 (lane 4), the fourth polypeptide of 25.6 kDa (p26) was detected, in addition to the three MTH1 polypeptides. Furthermore, radioactivities of each band are almost even, indicating that each polypeptide is synthesized with equal efficiency.
Figure 4. In vitro transcription/translation products of ATG mutated cDNA templates. Type 2B (GC) cDNA mutants, designated 1m, 2m and 3m, were produced by replacing each uAUG with AUC. T7 transcripts of type 2B (GC) cDNA (WT) and its mutants were translated in vitro. Lanes 1 and 8, molecular weight marker; lane 2, no template cDNA; lane 3, pET8c-MTH1 cDNA; lane 4, type 2B (GC) MTH1 cDNA; lane 5, 1m template; lane 6, 2m template; lane 7, 3m template.
The results shown above strongly suggest that the four or three AUGs (AUG1-4) in B-type MTH1 mRNAs are simultaneously utilized as translation initiation codons, at least in the rabbit reticulocyte lysate. To confirm that production of the three larger MTH1 polypeptides are indeed initiated from the three uAUG codons (AUG1-3), each ATG codon in type 2B (GC) MTH1 cDNA was replaced with an ATC codon by means of site-directed mutagenesis. mRNAs prepared from these cDNAs were designated as 1m (AUC1), 2m (AUC2) and 3m (AUC3), and were subjected to in vitro translation (Fig. 4, lanes 5-7). In the case of 1m, which lacks AUG1, synthesis of only the largest p26 was abolished. When 2m or 3m were translated, synthesis of the p22 or p21, respectively, was exclusively abolished.
As any substitution of each AUG with AUC codon did not affect amounts or mobilities of the other polypeptides, except the corresponding one, we concluded that translation of the four MTH1 polypeptides is alternatively initiated from the four AUGs (AUG1-4) in the type B (GC) mRNA.
Polymorphic alteration in exon 2c of MTH1 gene determines the number of MTH1 polypeptides produced in vivo
To determine whether the polymorphic alteration in exon 2c of MTH1 gene indeed affects the expression of MTH1 polypeptides in vivo, we collected peripheral blood lymphocytes orlymphoblastoid cell lines established from healthy volunteers. As shown in Figure 5A, genomic DNA fragments containing exon 2 were amplified by PCR, and nucleotide sequences were determined by direct sequencing. Samples from three individuals (TORISU, Oh and HO), with GT/GT, GT/GC and GC/GC sequences at the polymorphic site, respectively, were selected for the following experiments (Fig. 5B).
A, B
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C, D
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Figure 5. Analysis of lymphoblastoid cell lines that show a different zygosity at the polymorphic site of exon 2c. (A) Strategy for genotyping of MTH1 allele of exon 2. Boxes represent exon 2. The genomic nucleotide sequence around exon 2 is shown. The star shows the polymorphic site. To amplify the entire region for the MTH1 exon 2, PCR of genomic DNA was done using primers 668-5 and 491-3. Purified PCR products were subjected to direct sequencing by Dye Terminator Cycle Sequencing, using primer GT-GC2. (B) Nucleotide sequences of three cell lines (TORISU, Oh, HO). Boxed dinucleotides correspond to GT or GC of the complementary strand. The genotype determined by electropherogram is shown below. (C) Type-specific RT-PCR of MTH1 mRNA. Total RNAs of three cell lines were reverse-transcribed with primer MTH-P. Amplification of specific 5[prime] region for each type of MTH1 mRNA was done. The PCR products were analyzed by 8% PAGE. Numbersabove each lane (1-4) correspond to types of 5[prime] primers used (T1-T4). PCR products indicated by arrow-heads are derived from MTH1 mRNAs, as confirmed by Southern blotting (36). Closed arrowheads indicate PCR products derived from type 1 and A-type (2A, 3A and 4A) mRNAs. Open arrowheads indicate those from B-type (2B, 3B and 4B) mRNAs. (D) Western blotting of three cell lines (TORISU, Oh and HO). Whole-cell extracts (1 × 106) were subjected to SDS-PAGE and to western blotting.
To confirm the effects of polymorphism on the alternative splicing of MTH1 transcripts, type-specific RT-PCR, in which a specific primer (T1-T4) for the unique 5[prime] region of each type of mRNA was used in combination of the 3[prime]-common primer (MTH2-17) (36), was performed using total RNA prepared from these cells (Fig. 5C). In any sample, a single cDNA fragment was almost equally amplified with the specific primer T1 for the type 1 transcript. With primers (T2, T3 and T4) for type 2, 3 and 4 transcripts, two cDNA fragments corresponding to each transcript of the subtype A and B were amplified in samples from TORISU (GT/GT) and Oh (GT/GC), but only the larger cDNA fragments derived from each subtype B transcripts were amplified in the sample from HO (GC/GC). In samples from the GT homozygote, cDNA fragments derived from subtype A transcripts are more abundant than those from subtype B (Fig. 5C, TORISU lanes 2-4). However, in the GT/GC heterozygote, both of the two cDNA fragments were amplified to a similar extent (Fig. 5C, Oh lanes 2-4). Thus, the polymorphism affects cellular contents of each MTH1 transcript as well as the pattern of alternative splicing of the transcripts.
MTH1 proteins expressed in these cells were examined by western blotting with the anti-MTH1 (Fig. 5D). In the GT/GT homozygote (TORISU), three bands corresponding to p22, p21 and p18 were detected, as seen in Jurkat and HeLa cells (Fig. 5D, lane 1). While, in the GT/GC heterozygote (Oh) and GC/GC homozygote (HO), a band corresponding to the p26 was detected in addition to the three polypeptides seen in the GT homozygote. Furthermore, expression levels for p26, p22 and p21 are higher in the GC homozygote than in the heterozygote which expresses more p22 and p21 than in the GT homozygote, presumably reflecting the increased copy numbers of B-type MTH1 mRNAs.
Linkage disequilibrium between the two polymorphisms of MTH1 gene
Apart from the polymorphism at exon 2 of MTH1 gene, we reported another polymorphic site in the exon 4 that corresponds to the codon 83 for the p18 MTH1 protein. There are two types of variants, one has a GTG83 codon and encodes the Val83-MTH1, and the other has an ATG83 codon encoding the Met83-MTH1 (38,40). The p18 form of Met83-MTH1 expressed in E.coli is more thermolabile than the other, with both its secondary structure and 8-oxo-dGTPase activity (38). As shown in Table 1, we examined distribution of this polymorphism in the Japanese population, living in Japan, including 400 healthy volunteers and 601 patients with various diseases. Allele frequencies of Val83 and Met83 in the healthy volunteers were 0.91 and 0.09, respectively. There were three homozygotes for Met83/Met83 out of 400 healthy volunteers, and the frequency (0.75%) agrees with the expected one based on the Hardy-Weinberg rule. On the other hand, we found eight homozygotes for Met83/Met83 out of 601 patients with hepatocellular carcinoma, lung cancer, psoriasis, tuberculosis and Parkinson's disease, and there is no statistically significant difference in the frequency of Met83/Met83 (1.33%) in the patient group than the frequency in the healthy volunteers. It may be noteworthy that two of 20 women patients (10%) with hepatocellular carcinoma were Met83 homozygotes, and that no Met83 homozygotes were found in healthy volunteers (144 cases) over 50 years of age, while seven Met83 homozygotes were found in the patient group (491 cases), all over 50 years of age.
Table 1. Distribution of Val83 and Met83 alleles of MTH1 in Japanese subjects
| Group | Case | MTH1 genotype [case (%)] | ||
| Val83/Val83 | Val83/Met83 | Met83/Met83 | ||
| Healthy volunteers | 400 | 330 (82.5%) | 67 (16.8%) | 3 (0.8%) |
| Expecteda | (82.6%) | (16.6%) | (0.8%) | |
| Hepatocellular carcinoma | 104 | 81 (77.9%) | 21 (20.2%) | 2 (1.9%) |
| Lung cancer | 186 | 155 (83.3%) | 29 (15.6%) | 2 (1.1%) |
| Psoriasis | 145 | 122 (84.1%) | 21 (14.5%) | 2 (1.4%) |
| Tuberculosis | 66 | 52 (78.8%) | 14 (21.2%) | 0 (0.0%) |
| Parkinson's disease | 100 | 83 (83.0%) | 15 (15.0%) | 2 (2.0%) |
| Total | 601 | 493 (82.0%) | 100 (16.6%) | 8 (1.3%) |
aExpected frequency for each genotype was calculated from allele frequencies among healthy volunteers, as based on Hardy-Weinberg distribution.
Western blotting analysis of samples from tumor tissues surgically resected from lung cancer patients revealed that two of the Met83 homozygotes but not any Val83 homozygotes express the four MTH1 polypeptides, p26, p22, p21 and p18, as seen in the GC/GC homozygote (our unpublished data). Thus, it is likely that there is linkage between the two polymorphisms of MTH1 gene. To examine this possibility, we determined genotypes for the polymorphism at the exon 2c in all the Met83 homozygotes we found. Ten of 11 Met83 homozygotes were also homozygous for the GC polymorphism at exon 2c, and one was a heterozygote of GT/GC, indicating that 95% of Met83 alleles are linked to the GC allele (Table 2). Next we determined polymorphic alterations at the exon 2c in 12 individuals randomly selected from the population listed in the Table 1. As shown in the lower part of Table 2, 10 individuals who are homozygous for Val83 are also homozygous for the GT polymorphism, and the other two are compound heterozygotes with Val83/Met83 and GT/GC. Thus, there is an apparent linkage between Met83 and GT or Val83 and GC polymorphisms, respectively.
Table 2. Linkage of two polymorphic alterations in MTH1 gene
| Polymorphism at codon 83 for p18 | Polymorphism at the beginning of exon 2 | ||
| GT/GT | GT/GC | GC/GC | |
| Met83/Met83 | - | 1 | 10 |
| Val83/Val83 | 10 | - | - |
| Val83/Met83 | - | 2 | - |
DISCUSSION
Multiple translation products of MTH1 mRNAs by alternative translation initiation
Based on the following evidence, we concluded that human cells indeed produce three or four MTH1 polypeptides in vivo, depending on their genotype. First of all, three polypeptides (p22, p21 and p18) reacting with the anti-MTH1 and anti-M78 antibodies were detected in Jurkat and HeLa cells, the molecular weights being identical to those produced by in vitro translation of MTH1 mRNAs prepared from cDNA clones derived from Jurkat cells. Secondly, cells derived from the GC/GC homozygote which possess only type 1, 2B, 3B and 4B mRNAs, or cells from GT/GC heterozygote express an additional 25.6 kDa polypeptide (p26) reacting with the anti-MTH1, as seen in in vitro translation of the corresponding MTH1 mRNAs. Furthermore, cells derived from the heterozygote carry more B-type MTH1 mRNAs than those from the GT/GT homozygote, and produce more p22 and p21 MTH1 polypeptides.
These genetic and immunological findings support our conclusion. However, there is the possibility that MTH1 polypeptides may be processed or modified in vivo after translation. As shown in Table 3, each MTH1 protein is expected to have a unique N-terminal sequence, therefore determination of N-terminal sequences of these proteins, or detection with specific antibodies against each N-terminal sequence might be necessary to reveal the true features of these MTH1 proteins. We are now raising specific antibodies against each N-terminal region.
Table 3. Nucleotide sequences around alternative AUG codons and N-terminal sequences of the four MTH1 polypeptides
| Initiation site | Nucleotide sequence around initiation codona | N-terminal amino acid sequences |
| AUG1 | CCUUGAUGUAC | MYWSNQITRRLGERVQGFMSGISPQQMGEPEGSWSGKNPGTMGASR |
| AUG2 | GUUUUAUGAGU | MSGISPQQMGEPEGSWSGKNPGTMGASR |
| AUG3 | AGCAGAUGGGG | MGEPEGSWSGKNPGTMGASR |
| AUG4 | GGACCAUGGGC | MGASR |
In vertebrates, sequences surrounding an AUG codon determine the efficiency with which AUG functions as an initiation codon for translation. Kozak demonstrated that an AUG with purine at the -3 position and guanine at +4 position (the A in the AUG codon is designated +1) is efficiently utilized as the site of initiation. Sequences surrounding the four AUG codons in MTH1 mRNAs are shown in Table 3. The sequence surrounding the AUG4 is fairly consistent with Kozak's consensus (41). In contrast, sequences around AUG1 and AUG2 do not match the consensus. In AUG3, guanine is present at +4, but purine is not at -3. While AUG1, AUG2 and AUG3 of B-type mRNAs are not in the optimal context, our results of in vitro translation indicate that translation from each AUG occurs with an almost equal efficiency. Judging from relative contents of each MTH1 transcript and MTH1 polypeptides in cells, it is likely that translation initiation from each AUG in the B-type or even from AUG4 in the type 1 and A-type transcripts occurs with a similar efficiency.
For translation initiation in eukaryotic cells a model, called the `scanning model', in which the 40S ribosomal subunit with its associated factors engages an mRNA at or near its 5[prime]-cap and then scans in a 3[prime] direction, has been proposed (42). If nucleotides flanking the first AUG codon are suboptimal for initiation, some 40S ribosomal subunit may fail to recognize the AUG and continue scanning the next AUG codon. This mechanism is called `leaky scanning'. Several eukaryotic genes, for example mouse LAP (43), int-2 (44), mouse GATA-1 gene (45) and human nifs gene (46), express two or more proteins from alternative in-frame AUGs. In the case of the MTH1 gene, p18 MTH1 protein is expected to be translated from all types of MTH1 mRNAs; however, two or three MTH1 proteins (p26 and/or p22 and p21) must be translated from B-type mRNAs, which have three suboptimal initiation codons. Hence, it is likely that these initiation codons are utilized alternatively.
Functions of multi-forms of MTH1 proteins
As summarized in Table 3, each four-MTH1 polypeptide has a unique N-terminal sequence. Since these proteins may have alternative, cooperative, or even opposing activities (42), they would provide a significant consequence for MTH1 gene function. We purified and examined 8-oxo-dGTPase activity of each four recombinant MTH1 proteins expressed in E.coli, and found that all the MTH1 preparations have an equivalent activity to hydrolyze 8-oxo-dGTP (H.Oda, H.Hayakawa and Y.Nakabeppu, unpublished data). These results suggest that MTH1 polypeptides may function cooperatively in human cells. It has been shown that E.coli MutT protein and human p18 MTH1 protein also hydrolyze 8-oxo-GTP, and thus prevent misincorporation of 8-oxo-GTP into mRNA by RNA polymerase (31,47). We analyzed the activity of each MTH1 protein to hydrolyze 8-oxo-GTP, and found again that all have a similar level of activity. Recently, we found that MTH1 (p18) hydrolyzes oxidized forms of dATP, 2-hydroxy-dATP and 8-oxo-dATP as well as 8-oxo-dGTP(48), but MutT does not hydrolyze the two oxidized forms of dATP. Among the four MTH1 proteins, there may be differences in substrate specificities towards these oxidized nucleotides.
Subcellular localization of MTH1 proteins
It has been shown that targeting of a human iron-sulfur cluster assembly enzyme, nifs, to different subcellular compartments is regulated through alternative AUG utilization (46). Thus, different N-terminal sequences in the four MTH1 polypeptides may also function as signals for transport to different subcellular compartments. We reported earlier that most of the 18 kDa MTH1 protein is localized in the cytoplasm, and ~5% is in the mitochondrial matrix (37). Since the mechanism of MTH1 protein transport into mitochondria has not been elucidated, we analyzed the probability of each MTH1 polypeptide for mitochondrial targeting, using MitoProt Program II (49). Results suggest that p26 and p18 can be imported into mitochondria, and the former is likely to have a much better mitochondrial targeting signal than does the latter, a part of which proved to be imported into mitochondria (37). Preliminary data show that the 18 amino acid leader sequence of p26 MTH1 indeed functions as the mitochondrial targeting signal when fused to the green fluorescent protein (V.Y.Sakai and Y.Nakabeppu, personal communication).
Furthermore, we demonstrated an exclusive linkage between two polymorphic sites, Met83 and GC at exon 2c, or Val83 and GT at exon 2c. Met83-MTH1(p18) was demonstrated to be less thermostable than was Val83-MTH1(p18), both in its 8-oxo-dGTPase activity and secondary structure (38). The exclusive linkage results in synthesis of Met83-MTH1(p26) but not Val83-MTH1(p26). One of the possibilities for this linkage might be attributed to a deficiency in Met83-MTH1 polypeptides (p22, p21 and p18) which can be overcome with the unique leader sequence found in the p26 MTH1 polypeptide. To evaluate this notion, subcellular localization of each MTH1 polypeptide has to be determined.
If each MTH1 isoform has a distinct function or different intracellular localization, it might also be interesting to investigate whether there is a change in the ratio of the isoforms under conditions where expression levels of some translation initiation factors, such as eIF-4E or eIF-4G, are varied, especially in different tumor types, as reported for FGF-2 isoforms (50).
Genetic polymorphisms in MTH1 gene and their effects
Zygosity of the polymorphism at MTH1 exon 2c affects the amount of each MTH1 polypeptide as well as the number of MTH1 polypeptides synthesized. It is noteworthy that p26 MTH1 polypeptide translated from AUG1 exists only in individuals who have a haplotype (GC and Met83) of the MTH1 gene, withsome exception. Amounts of p22 and p21 are also dependent on dosages of the GC allele. A more detailed analysis on physiological effects and distribution of these polymorphisms among various human populations is needed.
ACKNOWLEDGEMENTS
We extend special thanks to Drs H. Hayakawa and M. Furuichi for helpful discussions, Drs T. Tana and T. Sasazuki for providing lymphoblastoid cell lines, Drs K. Sugio, K. Takenaka and K. Sugimachi for surgical specimens, Drs Y. Mizuno and T. Kondo for DNAs from patients of Parkinson's disease, and M. Ohara for comments on the manuscript. This work was supported in part by Kyushu University Interdisciplinary Programs in Education and Projects in Research Development (Y.N.) and a Grant-in-Aid for Scientific Research to H.O. (10770233) and to Y.N. from the Ministry of Education, Science, Sports and Culture of Japan.
REFERENCES
*To whom correspondence should be addressed at: Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan. Tel: +81 92 642 6800; Fax: +81 92 642 6791; Email: yusaku{at}bioreg.kyushu-u.ac.jp
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