Identification of a truncated form of methionine sulfoxide reductase a expressed in mouse embryonic stem cells
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Methionine Sulfoxide Reductase A (MsrA), an enzyme in the Msr gene family, is important in the cellular anti-oxidative stress defense mechanism. It acts by reducing the oxidized methionine sulfoxide in proteins back to sulfide and by reducing the cellular level of reactive oxygen species. MsrA, the only enzyme in the Msr gene family that can reduce the S-form epimers of methionine sulfoxide, has been located in different cellular compartments including mitochondria, cytosol and nuclei of various cell lines.
In the present study, we have isolated a truncated form of the MsrA transcript from cultured mouse embryonic stem cells and performed eGFP fusion protein expression, confocal microscopy and real time RT-PCR studies.
Results show a different expression response of this truncated transcript to oxygen deprivation and reoxygenation treatments in stem cells, compared to the longer full length form. In addition, a different subcellular localization pattern was noted with most of the eGFP fusion protein detected in the cytosol.
One possibility for the existence of a truncated form of the MsrA transcripts could be that with a smaller protein size, yet retaining a GCWFG action site, this protein might have easier access to oxidize methionine residues on proteins than the longer form of the MsrA protein, thus having an evolutionary selection advantage. This research opens the door for further study on the role and function of the truncated MsrA embryonic mouse stem cells.
KeywordsMouse Embryonic Stem Cell Methionine Sulfoxide Reductase Methionine Sulfoxide Anoxia Treatment eGFP Fusion Protein
Free radical damage of cellular components, including proteins, has long been recognized in physiological aging and disease conditions. One of the cellular defense mechanisms to reduce the oxidized residues in proteins, and thus restore their functions as well as reduce oxidative stress, relies on the methionine sulfoxide reductase (msr) family genes, of which three MsrB genes (MsrB1, B2 and B3), have been identified in mammals [1, 2, 3, 4, 5, 6, 7]. Only one MsA gene has been found in mammals [1, 2, 3, 4, 5, 6, 7]. While both MsrA and MsrBs conduct the redox reactions with a similar chemical reaction mechanism, MsrBs can convert methionine-R-oxide (R-MetO) back to methionine while MsrA reduces methionine-S-oxide (S-MetO), respectively[4, 8]. MsrBs localize at different cellular compartments: 1. MsrB1 is a cytosolic and nuclear protein; and 2. MsrB2 is targeted to mitochondria. Human MsrB3 gives rise to two forms generated by alternative first exon splicing, which are targeted to the endoplasmic reticulum (ER) and mitochondria  of the cell.
Although only a single MsrA gene is found in mammals, the corresponding protein is found to localize in multiple cellular compartments . Further studies on human MsrA gene structures have identified two distinct putative promoters that generate three transcripts. The main MSRA transcript (MsrA1) was translated into the longest protein which localizes in mitochondria. MsrA2 and 3 originate from a second promoter and target the cytosol and nuclei [10, 11]. More recent studies from rat smooth muscle cells revealed two novel splice forms: MsrA2a and MsrA2b . The alternative splicing event occurred at the level of the second exon with MsrA2a coding a functional isoform. It seems that both alternative promoters and alternative splicing contribute to the variety of MsrA isoforms that are responsible for methionine sulfoxide reduction in different cellular compartments.
To date, most of the studies on MsrA isoforms are focused on the 5' terminus where a mitochondrial signal peptide is alternatively presented in different isoforms dictating whether or not the protein products are localized in the mitochondria [10, 12, 13]. Although there is evidence that transcripts of MsrA from alternative splicings at the 3' end of the MsrA gene are present in the mammalian EST database, due to the concern that these transcripts might not translate into enzymatically active protein products, no detailed studies to date have been reported on these transcripts .
We have consistently found a MsrA transcript from alternative splicing at the 3' end, skipping exon 5, thus producing a shorter isoform with a presumably truncated protein product containing the conserved catalytic active site in cultured mouse embryonic stem cells. Due to the importance of anti-oxidative stress mechanisms in stem cells, we have performed studies on this isoform in its expression pattern in normal culture conditions and its response to oxygen depletion/reoxygenation conditions in mouse embryonic stem cells.
Mouse embryonic stem cell culture
The mouse embryonic stem (MES) cells (CCE-24) were routinely grown on 0.1% gelatin-coated dishes in Dubecco's Modified Eagle's medium (DMEM) containing 15% heat-inactivated fetal bovine serum (catalog # 10100, Invitrogen, Carlsbad, CA), 10 ng/ml human leukemia inhibitory factor (LIF) (LIF2010, Millipore, Billerica, MA), and monothioglycerol (Sigma, St. Louis, Mo.) at 4.5 × 10-4 M . Cells were grown on tissue culture plates coated with 0.1% gelatin (Sigma, St. Louis, WA) and routinely split every two days at 1:4 to 1:10 and immunostained for stem cell specific markers SSEA-1 (Mab4301) and SSEA-4 (Mab4304, Millipore, Billerica, MA) to ensure no differentiation. Only cells within the 20th passage were used.
The anoxic treatment of mouse embryonic stem cells was performed by incubating the cells in an anaerobic chamber (Sheldon Manufacturing Inc., Cornelius, OR) supplied with 90% nitrogen gas, 5% hydrogen gas and 5% carbon dioxide at 37°C. Cells were removed after selected periods of treatment and incubated again in a regular cell culture incubator at 37°C for designated times.
Cloning of the truncated form of MsrA cDNA and construction of MsrA-truncated-eGFP fusion expression plasmid followed methods routine in our laboratory and recently published .
MsrA cDNA was amplified from the total RNA extracted from cultured embryonic stem cells using the primer pairs of: MsrA-for: 5'-cctggctgcggaggtggagaaac and MsrA-rev: 5'-atggccatcgggcaggaaactcc. The 744bp DNA band was gel purified and ligated into pGEM-T easy vector (Promega, WI). After sequencing to confirm gene sequences and rule out mutations, PCR was performed again using the following primer pairs to amplify the truncated form of MsrA and introduce a BamHI cutting site at the 3' end (MsrA-for: 5'-cctggctgcggaggtggagaaac and MsrA-BamHI-rev: 5'-tggggccaaggatccgctttgaaagaacc). The amplicons were gel purified and ligated into pEGFP-C vectors (Invitrogen, CA) to construct the truncated MsrA-eGFP expression plasmid. The truncated MsrA cDNA in the final plasmid was sequenced again to confirm the open reading frame.
After the MsrA-truncated-eGFP fusion expression plasmid was transfected into cells for three to five days and after confirmation of the GFP fluorescence signal by epi-fluorescent microscopy, 500 nM Mitotracker (Molecular Probe (Invitrogen), Carlsbad, CA) was diluted with complete culture medium and added to the cells. The cells were incubated for 30 minutes and checked under epi-fluorescence microscopy to confirm that they were well stained by the dye. The Mitotracker medium was discarded. Cells were washed with PBS and fixed in 1% Formaldhyde in PBS for 15 minutes at room temperature. Cells were rinsed with PBS twice and incubated in a 1/10000 dilution solution of DAPI (10 mg/ml) in PBS for 15-20 minutes. Cells were then mounted in the mounting solution Pro-Long (Molecular Probe, Carlsbad, CA), air-dried for 2 days in the dark, and coverslips were sealed to the slides with fingernail polish. Confocal microscopy was carried out using a Carl Zeiss confocal microscope at the University of Miami Diabetes Research Institute. Five micron Z-series were scanned for each sample.
Real time RT-PCR
MsrA-long form-for: 5'-TCTGGGTCTTGAAAGGAGTGTA;
MsrA-long-form-rev: 5'-AGGTATTGCTGGTGGTAGTCTTC; Amplicon size: 395bp.
MsrA-truncated-rev: 5'-GCCATCGGGCAGGAAACTCCAG; Amplicon size:168bp
β-actin-for: 5'-CAGCAATGCCTGGGTACATGGTG; Amplicon size: 249bp.
Results and Discussion
Evidence of the existence of a smaller form of MsrA in mouse embryonic stem cells
Cloning of the cDNA of the truncated form of MsrA
Confocal microscopy reveals different subcellular localizations for the truncated MsrA protein compared to the full length using eGFP fusion constructs
Recent studies from Lee et al.,  and Pascual et al.,  have demonstrated the existence of two alternative promoters for the MsrA gene that encodes different isoforms of MsrA proteins that locate in mitochondria or cytosol/nuclei due to the presence or absence of a N-terminal mitochondrial signal peptide. However, studies from Kim and Gladyshev , using GFP fusion techniques and deletion mutagenesis have revealed other important functional domains in the MsrA peptide sequence, including sequences close to the c-terminus, that may also direct the specific locations of the protein in subcellular compartments. In addition, localization of the mitochondrial form of MsrA in the cytosol and nuclei is also noted by Kim and Gladyshev  in MsrA overexpression studies. Although syntheses of different isoforms with or without N-terminal signal peptide might be the optimal way for the cells to direct protein sorting, it definitely should not be ignored that the same isoform might still be able to locate to multiple cellular compartments. While we do not rule out the possibility that the altered localization pattern for the truncated protein compared to the long form is due to GFP fusion interference, it is most unlikely considering the fact the same method has been used successfully to reveal subcellular localization of MsrA in our cell lines . Our studies on the truncated MsrA-eGFP fusion protein suggest a necessary domain at the c-terminal sequence for permanently docking of the protein on mitochondria. In addition to the mitochondria signal peptide, there might exist another essential domain at the c-terminal end of the full length protein, without which, the truncated proteins, are able to be sorted to the mitochondria but will eventually leak out back into the cytosol. In the deletion mutagenesis studies of Kim and Gladyshev , the deletion is limited only to the very end of the N-terminus or very middle, not totally overlapping the portion omitted in this truncated form which could harbor more functional domain units .
Real time RT-PCR shows a different response of mRNA expression levels for the trunctated form compared to the full length MsrA
In summary, a c-terminal truncated form of MsrA has been cloned from mouse embryonic stem cells due to the skipping of exon 5 and subsequent frame shift in exon 6, generating a premature stop codon. The truncated protein shows a different subcellular localization and pattern of expression response to anoxia/reoxygenation treatment on the stem cells. Further study on the enzymatic activity of this peptide is needed to consider it as a functional isoform. One possibility for the necessity of having such a truncated form could be that with a smaller protein size and retained GCWFG active site, this protein might have easier access to the oxidized methionine residues on proteins with structure hindering the access for the long form protein, thus having an advantage to be evolutionarily selected and maintained. Since the truncated protein itself does not contain the c-terminal cysteines, whether the final relieving of the oxidation step needs the long form MsrA is unknown, although we did observe that heterodimers formed between long form and truncated proteins in Western blotting experiments.
The study was funded by NIH grants HL-58435 and HL-61246 and an American Heart Association Grant to LFL, an American Heart Association grant to X.H., an NIH grant to K.A.W. and a NIH Summer Research Scholarship to C.Z. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, lung and Blood Institute of Health. We are grateful to Mrs. Amy Patrick for outstanding secretarial and administrative assistance in the preparation and submission of this manuscript.
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