Molecular Characterization of Chinese Hamster Cells Mutants Affected in Adenosine Kinase and Showing Novel Genetic and Biochemical Characteristics
© Cui et al; licensee BioMed Central Ltd. 2011
Received: 16 November 2010
Accepted: 17 May 2011
Published: 17 May 2011
Two isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells. However, there is no information available regarding the unique functional aspects or regulation of these isoforms.
We show that the two AdK isoforms differ only in their first exons and the promoter regions; hence they arise via differential splicing of their first exons with the other exons common to both isoforms. The expression of these isoforms also varied greatly in different rat tissues and cell lines with some tissues expressing both isoforms and others expressing only one of the isoforms. To gain insights into cellular functions of these isoforms, mutants resistant to toxic adenosine analogs formycin A and tubercidin were selected from Chinese hamster (CH) cell lines expressing either one or both isoforms. The AdK activity in most of these mutants was reduced to <5% of wild-type cells and they also showed large differences in the expression of the two isoforms. Thus, the genetic alterations in these mutants likely affected both regulatory and structural regions of AdK. We have characterized the molecular alterations in a number of these mutants. One of these mutants lacking AdK activity was affected in the conserved NxxE motif thereby providing evidence that this motif involved in the binding of Mg2+ and phosphate ions is essential for AdK function. Another mutant, FomR-4, exhibiting increased resistance to only C-adenosine analogs and whose resistance was expressed dominantly in cell-hybrids contained a single mutation leading to Ser191Phe alteration in AdK. We demonstrate that this mutation in AdK is sufficient to confer the novel genetic and biochemical characteristics of this mutant. The unusual genetic and biochemical characteristics of the FomR-4 mutant suggest that AdK in this mutant might be complexed with the enzyme AMP-kinase. Several other AdK mutants were altered in surface residues that likely affect its binding to the adenosine analogs and its interaction with other cellular proteins.
These AdK mutants provide important insights as well as novel tools for understanding the cellular functions of the two isoforms and their regulation in mammalian cells.
Adenosine kinase (AdK) is a major purine salvage pathway enzyme belonging to the ribokinase family of proteins [1–4]. It plays a central role in regulating the intracellular and interstitial concentrations of the purine nucleoside adenosine (Ado), which exhibits potent cardioprotective and neuroprotective activity [5–7]. During ischemia, the compromised regeneration of ATP causes an increase in the intracellular concentration of Ado, which results in its net efflux into extracellular space where it binds to Gi/o-coupled Ado receptors: A1, A2A, A2B, and A3, to modulates a variety of physiological responses to reduce tissue damage from ischemic injury [5, 6, 8–10]. The expression of AdK undergoes rapid coordinated changes in the brain following epileptic seizures or stroke, resulting in an acute surge of Ado, which serves to minimize damage to the brain [6, 11]. Strong evidence in support of the protective role of Ado has been obtained from studies where transient down regulation of AdK after acute brain injury protected brain from seizures and cell death, whereas its overexpression as in epilepsy caused seizure aggravation and promoted cell death [11–13].
AdK, in addition to its central role in purine salvage and ATP catabolism, also plays a critical role in the maintenance of methylation reactions. In the S-adenosylmethionine (SAM) dependent methylation pathway, Ado and homocysteine (Hcy) are produced as a result of hydrolysis of S-adenosyl-homocysteine (SAH), which is the common end product of all methylation reactions [1, 14–17]. The hydrolysis reaction, which is catalyzed by the enzyme SAH-hydrolase, is reversible and the equilibrium constant of this reaction favors SAH formation. Hence, unless the hydrolysis product, Ado and Hcy are rapidly removed, it will lead to the buildup of SAH, which is a potent inhibitor of transmethylation reactions [14, 17, 18]. In the guinea-pig heart, the transmethylation pathway has been shown to be an important intracellular source of Ado under normal conditions and the Ado produced by this mechanism is mainly salvaged by AdK . Studies with the AdK knockout mouse, which causes liver failure and early postnatal death , indicate that the effects of AdK deficiency on transmethylation reactions are the main underlying causes for its lethal effect . The deficiency of AdK due to its pivotal role in the maintenance of transmethylation reaction also causes developmental abnormalities and reduced salt stress in plants [20, 21].
Two isoforms of AdK are present in mammalian species [22–25]. These isoforms differ from each other only in their N-termini. The long isoform (AdK-L) of AdK contains an extra 20-21 amino acids in place of the first four amino acids of the AdK-short (AdK-S) isoform [23, 26]. Studies with the recombinant AdK-L and AdK-S proteins have revealed no differences in their biochemical or kinetic properties (unpublished results). However, we recently showed that the N-terminal extension in the AdK-L functions as a nuclear localization signal . Thus, of the two AdK isoforms, AdK-L is targeted to the nucleus whereas AdK-S is localized in the cytoplasm . The differential subcellular localization of these two AdK isoforms suggests that they carry out different physiological functions. However, there is no information available at present regarding the unique cellular functions of these isoforms or how their expression is regulated.
Unlike the lethal phenotype of AdK-/- mice, AdK deficient mutants can be readily obtained in cultured cells by selecting in the presence of toxic concentrations of the Ado analogs [25, 28–31]. Most of such mutants lack AdK activity and some mutants that have been studied in detail contained large deletions within the AdK gene [24, 32]. Our recent work shows that in contrast to the Chinese Hamster Ovary (CHO) cells that expresses only the AdK-L isoform, in the CH V79 cell line established from embryonic lung  both AdK-L and AdK-S isoforms are expressed. Hence, to gain further insights into the cellular functions of AdK, in the present work we report the isolation and characterization of mutants resistant to Ado analogs from V79 and other CH cell lines. Our results show that these mutants exhibit interesting differences in their cross-resistance pattern towards the N- and C- Ado analogs and also in the expression profiles of the two AdK isoforms. (Note: In N-nucleosides the purine base is linked to ribose via a N-C bond, whereas in C-nucleosides this linkage involves a C-C bond [34, 35]). Several of these mutants contained novel molecular alterations in AdK affecting its activity/function. One of these mutants that exhibited increased resistance to only C-Ado analogs, and whose drug-resistance phenotype was dominantly expressed in cell hybrids formed with AdK+ cells, has been characterized in detail . We showed that a single point mutation in AdK is responsible for its novel genetic and biochemical characteristics. These mutants provide important insights and novel tools for understanding the cellular functions and regulation of the AdK isoforms in mammalian cells.
The AdK-L and AdK-S Isoforms Differ in their First Exons and the Promoter Sequences
Expression of the Two Isoforms Differ in Various Tissues and Cell Lines
Isolation and Characterization of Mutants Resistant to Adenosine Analogs from V79 Cells
To gain insights into the cellular functions of AdK and the regulation of its isoforms, mutants resistant to the Ado analogs tubercidin and formycin A (FoA) were selected from V79 cells. Of these analogs, tubercidin (and also toyocamycin) similar to adenosine is an N-nucleoside, whereas FoA is a C-nucleoside analog. In our earlier work, interesting differences have been observed in the cross-resistance patterns of AdK mutants towards the N- and C- Ado analogs [31, 34]. Under the conditions employed (see Methods section), the resistant mutants were obtained at a frequency of ~ 2.5 × 10-7.
Degree of Resistance of Mutants Cell Lines to Adenosine Analogs
V79 Cell Line
Relative Resistance of the Mutant Cell lines
1 (~20 ng/ml)
1 (~10 ng/ml)
Molecular Characterization of the Mutants affected in AdK
The mutants of CHO cells lacking in AdK activity that have been previously studied contained large deletions in the AdK gene  and they provided no useful information regarding structure-activity aspects of AdK. With the availability of a good antibody to AdK, the mutants lacking AdK antibody cross-reactive bands can now be readily identified and excluded from further analyses. These studies have revealed that the mutants DrToyR-18 and FomR-4 isolated in our earlier work both contained normal amounts of the AdK antibody cross-reactive bands (see Figure 3). Hence, these mutants and two other mutants VF18 and VF19 obtained in this work were further characterized.
The DrToyR-18 mutant is highly resistant to both N- and C- adenosine analogs. The sequencing of AdK cDNA from this mutant revealed that it contained a single point mutation (G → A substitution), which changes a conserved Glu242 residue in the NxxE motif to Lys (Figure 5A). Earlier studies indicate that the conserved Asn and Glu residues in the NxxE motif (boxed in Figure 5A) are involved in the binding of activating phosphate ion, indicating that this motif is essential for AdK function [1, 45]. We have also introduced the E242K mutation in CHO AdK cDNA in pET-15b expression vector . Upon expression, the resulting protein showed no AdK activity (results not shown) confirming that this mutation leads to the inactivation of the enzyme and is responsible for the drug resistance phenotype of the DrToyR18 mutant. These results provide strong evidence that the NxxE motif is essential for AdK function in vivo.
The AdK cDNA was also PCR sequenced from the VF18 and VF19 mutants. For VF18 mutant, of the four clones sequenced, 2 contained a single point mutation changing the Leu188 into Phe. The remaining two clones showed no change in the AdK sequence. Similarly, of the 4 clones that were sequenced from the VF19 mutant, 2 contained the same mutation (Leu188Phe) as observed in the VF18 mutant. However, these clones in addition also contained a second mutation changing Phe221 into Leu. The mutant VF19 displays a higher degree of resistance to FoA in comparison to the VF18. Thus, it is possible that these two mutations synergistically reduce the binding of FoA to AdK. The locations of various amino acids that are altered in these mutants i.e. Ser191Phe, Leu188Phe, Phe221Leu and Glu242Lys in the human AdK structure are shown in Figure 5 B and 5C.
Discussion and Conclusion
This paper presents information regarding several novel characteristics of AdK from mammalian cell lines. We show that the two isoforms of AdK differ from each other only in their first exons. Because each of these isoforms contains its own promoter, the transcription of these two isoforms should be regulated independently at the genetic level. The expression of the two isoforms also differs greatly in various rat tissues. Whereas in liver, kidney, lung and pancreas both isoforms were expressed at comparable levels, in other tissues such as heart, thymus, skeletal muscle and brain, either the AdK-L or AdK-S isoform is predominantly expressed. Large differences in the expression of these two isoforms have also been observed in earlier studies [12, 22, 23]. Importantly, our studies also showed that the expression of the two isoforms also differed greatly in established cell lines. Whereas the CHO and Hela cells expressed only the AdK-L isoform, in two other cell lines V79 and GM7S from the same species, both AdK isoforms were expressed in comparable amounts. It is of interest that the V79 and GM7 cell lines, which are derived from lung, expresses both isoforms. Therefore, it is possible that the expression of these isoforms in cultured cells reflect their expression profiles in corresponding tissues.
However, the main focus of this work was on isolation and characterization of mutant CH cell lines that are affected in AdK. From CH V79 cell line expressing both AdK isoforms, 24 mutants resistant to FoA and tubercidin were isolated. About half of these mutants were highly resistant to both tubercidin and FoA, whereas the remaining, although they were highly resistant to FoA, exhibited only marginal resistance to tubercidin. Biochemical studies have revealed that all of these mutants except VF18 and VF19 contained either no or very low level of AdK activity. This accounts for their resistance to the Ado analogs, which are converted into their toxic forms by AdK. The expression of the two AdK isoforms in different mutants also exhibited important differences. Whereas the mutants VF6, VF9, VT4 and VT5 containing only background AdK activity lacked both isoforms, many other mutants with similar level of AdK activity expressed either the AdK-S isoform viz. VF13, VF24 and VF26 or the AdK-L isoform viz. the mutants VF1-7 and VF12. Surprisingly, the mutants VF8, VF15, VF20 and VT2 that show negligible or greatly reduced level of AdK activity expressed both AdK isoforms at a level comparable to the WT V79 cells. Although, the molecular alterations in most of these mutants remain to be identified, it is quite likely that in mutants that do not express either of these isoforms, mutations or deletions affecting one or more of the common AdK exons (exons 2-11) have occurred. These mutants could be similar to the mutants CHO cells that have been previously characterized, which contained large deletions leading to loss of several introns and exons [32, 36]. In contrast to these mutants, the mutants where expression of either the AdK-L or AdK-S isoform is preferentially affected are most likely to contain mutations in the promoter regions for these isoforms. Thus, further molecular characterization of these mutants, such as the methylation status, should provide useful insights concerning the functional significance of the two isoforms and how their expression may be regulated in mammalian cells.
The molecular alterations in a number of mutants resistant to Ado analogs were also identified in the present studies. One of these mutants, DrToyR-18 was specifically altered in the conserved NxxE motif, which has been indicated to be important in the binding of activating phosphate ion to AdK as well as other PfkB family of proteins [1, 4, 45, 46]. The complete loss of AdK activity in this mutant, both in vivo and in vitro, now provides direct evidence that this motif, which is in close proximity to the binding sites for Mg2+ ion as well the substrate adenosine (Figure 5B), is essential for AdK function in vivo.
In this work, we have also identified the molecular alteration in the FomR-4 mutant, whose genetic and biochemical characteristics have remained puzzling for >25 years. We show that this mutant contains a single base substitution mutation that changes a conserved Ser191 into Phe. This mutation when introduced into either CH or human AdK (results not shown) was sufficient to confer a similar genetic and biochemical phenotype as observed for the FomR-4 mutant. The Ser191Phe mutation is present in the AdK structure  on the periphery of the protein near the entrance of the substrate-binding pocket (Figure 5C). The C- Ado analogs, to whom the FomR-4 mutant exhibits selective resistance exist predominantly in Syn conformation in contrast to the Anti-conformation for Ado and various N-nucleoside analogs [31, 35]. Hence, it is likely that this molecular alteration selectively prevents the binding of FoA and other C-nucleosides to the mutant enzyme.
However, there are two other puzzling aspects of the FomR-4 mutant that remain to be addressed. First, the cell extracts from this mutant show no AdK activity despite its containing AdK activity in vivo. Second, the drug-resistance phenotype of this mutant is dominant in cell hybrids formed with WT cells [34, 41]. The first observation suggests that the reaction product of AdK i.e. AMP is not released from the mutant enzyme under in vitro conditions, but in the cellular milieu it is likely directly transferred to the next enzyme AMP-kinase (AMPK) in the pathway. The AMPK, which carries out the reaction AMP + ATP ↔ 2 ADP, plays a key role in maintaining the equilibrium concentrations of all three adenine nucleotides . The subsequent phosphorylation of ADP into ATP is carried out by the enzyme nucleoside diphosphate kinase . Because the toxicity of Ado analogs (e.g. FoA) requires their phosphorylation into higher phosphorylated forms (e.g. di- and tri-phosphates), which interfere with different metabolic pathways [31, 35, 49], if the mutation in AdK prevents the conversion of FoA or FoA-PO4 into higher phosphorylated forms then their toxicity will be averted.
Lastly, we have also identified the molecular alterations in two of the V79 cell mutants, V18 and VF19, which similar to the FomR-4 mutant also exhibit preferential resistance to the C-Ado analogs. The observed molecular alterations in these two mutants are located on the same face of AdK as the mutation in the FomR-4 mutant (Figure 5C). Thus, it is likely that the genetic changes in these mutants, Leu188Phe and Phe221Leu, also selectively affect the binding of FoA to the mutant AdK. However, it is of interest that of the four clones that were sequenced for these mutants, only two contained mutational alterations, whereas the other two showed no change. This is in contrast to the FomR-4 mutant, where all four sequenced clones contained the Ser191Phe mutation. Earlier work on CHO and V79 cells has provided evidence that the CHO cell line is functional hemizygous for many genetic loci including the AdK gene, whereas in V79 cells two functional copies of these genes were inferred [29, 50]. In this context, our observations that in the FomR-4 cells only the mutated form of AdK was found, whereas in mutant V79 cells both WT and the mutated forms of AdK were present support to this inference. The fact that the VF18 and VF19 mutants are resistant to FoA, despite their containing normal levels of AdK activity, indicates that the mutations in them are also expressing codominantly.
The mutants VF18 and VF19 are also of much interest, because in comparison to all other AdK mutants, they are the only mutants exhibiting enhanced AdK activity relative to the parental V79 cells. These mutants also show higher expression of the AdK-S isoform in comparison to the AdK-L isoform. These observations indicate that these mutants, in addition to the molecular changes that we have identified in this work, also contain additional genetic changes affecting the expression of the two isoforms. To understand the functional significance of different molecular alterations in these mutants, it is of much interest to further characterize AdK from these mutants at genetic, molecular and biochemical levels.
Cell Culture and Cell Lines
The origins of various CH cell lines CHO, V79 and GM7S used in this work have been described in earlier work [29, 39]. The ToyR-4, DrToyR-18 and FomR-4 mutants of CHO cells were also isolated and partially characterized in earlier work [24, 32, 34]. Of these ToyR-4 and DrToyR-18 mutants are highly resistant to both N- and C-Ado analogs. The mutant DrToyR-18 was obtained from the Dr-31 cell line, which is a different clone of the original CHO cell line . The ToyR-4 mutant has previously been shown to contain a large deletion in the AdK gene [24, 29, 32], whereas the genetic lesion in DrToyR-18 has not yet been identified. The FomR-4 mutant was selected using FoA and its various characteristics are described in earlier work [34, 41]. The cells were grown in monolayer culture at 37°C in alpha medium supplemented with 5% fetal bovine serum in 95% air - 5% CO2 atmosphere. For selection of mutants, V79 cells were treated with 300 μg/ml of the mutagen ethyl methanesulfonate for 18 hrs and then grown for 5 days . The mutants were selected by plating 5 × 105 cells/dish in multiple dishes in medium containing either 200-500 ng/ml FoA (+ 10 μg/ml of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine) or 80 ng/ml tubercidin. The mutant colonies were expanded and maintained by growth in non-selective medium. The mutants were named based on the selective drug. Thus, V79FomR and V79TubR (annotated as VF and VT) denote mutants isolated using FoA and tubercidin. The degree of resistance of the cell lines towards Ado analogs was determined by plating 200 and 500 cells in medium containing different concentrations of the analogs as in earlier work [29, 34]. After 7 days, the colonies were fixed, stained with 0.5% methylene blue and their numbers were counted. Assuming the number of colonies formed in the absence of any drug to be 100%, the relative plating efficiencies of cell lines in presence of different drug concentrations were determined. The D10 value represents the drug concentration that reduces plating efficiency of a cell line to 10% of that observed in the absence of any drug . The sources of various Ado analogs and other chemicals have been described in earlier work [29, 34].
Adenosine kinase activity assay, Western blotting, RT-PCR and Sequencing
AdK activity was measured as described previously using a radioactive assay involving conversion of [3H]-adenosine to [3H]-AMP [29, 45]. [2,8-3H]-Adenosine (40 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. For Western blot analysis, 40 μg of cell extracts from different cell lines were electrophoresed on 12% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). After transfer to nitrocellulose and blocking, the blot was reacted with 1:1000 dilution of a rabbit polyclonal antibody to human recombinant AdK raised in our lab . After washing the blot was reacted with 1:2000 dilution of anti-rabbit lgG conjugated to horseradish peroxidase and then developed using 4-Chloro-1-naphthol and H2O2. All of the experiments were repeated at least twice with very similar results. The quantification of the results for the rat tissue experiments (three independent experiments) was carried out using the NIH ImageJ software and the average intensity ± SD was calculated. For RT-PCR, total RNA was isolated from 1-2 confluent dishes using TRIzol (Invitrogen) as per the manufacturer's protocol. cDNA was generated using RevertAid™ H Minus First Strand cDNA Synthesis Kits (Fermentas) with oligo(dT)18 primers. Full length AdK sequence was amplified from the cDNA using the forward primer 5'-ATGGCAGCTGCTGAGGAGC-3' and reverse primer 5'-TCAGTGGAAGTCTGGCTTCTC-3' based on CH AdK sequence . The amplified fragments were cloned and sequenced using the M13 forward and reverse primers.
Mammalian Cell Transfection and In vitro Mutagenesis
The full-length AdK cDNA (long isoform) from CH was cloned in the mammalian cell expression vector pcDNA3.1 . The S191→F mutation in the CH (or human) cDNA was made using the 'Quickchange' site-directed mutagenesis kit (Stratagene) as described in earlier work [27, 45]. The transfection of the wild-type (WT) or the mutant ToyR-4 CHO cells with these plasmid DNAs was carried out using the Lipofectamin-2000 reagent (Invitrogen) as described in earlier work . Stable transfectants expressing these genes were obtained by growing the cells in presence of G-418 (650 μg/ml) for more than 1 month. The degree of resistance of these transformants for tubercidin and FoA was determined as described above.
This work was supported by a research grant (T-6177) from the Heart and Stroke Foundation of Canada.
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