Ischemic preconditioning inhibits over-expression of arginyl-tRNA synthetase gene Rars in ischemia-injured neurons.

The expression changes of Rars gene in ischemia-injured neurons were investigated by detecting its translational product arginyl-tRNA synthetase (ArgRS), and the inhibitory effects of ischemic preconditioning (IPC) on Rars gene were explored. Both IPC model and prolonged ischemia (PI) model were established by using the classic oxygen glucose deprivation (OGD) method. The primary cultured neurons were assigned into the following groups: the experimental group (IPC+PI group), undergoing PI after a short period of IPC; the conditional control group (PI control group), subjected to PI without IPC; blank control group, the normally cultured neurons. The Rars transcriptional activities and ArgRS expression levels were measured at different time points after re-oxygenation (3 h/6 h/12 h/24 h). Data were collected and statistically analyzed. Compared to the blank control group, the Rars activities and ArgRS levels were significantly increased in PI control group, peaking at the time point of 6 h after re-oxygenation. Rars activities and ArgRS levels were significantly lower in the experimental group than in the PI control group at different time points after re-oxygenation. PI insult can induce an escalating activity of Rars and lead to ArgRS over-expression in primary cultured neurons. IPC can inhibit the increased Rars activity and down-regulate ArgRS expression of ischemia-insulted neurons. This mechanism may confer ischemic tolerance on neurons.

Expression of arginyl-tRNA synthetase in rats with focal cerebral ischemia.

Aminoacyl-tRNA syntheses (AARS) can catalyze the adenosine triphosphate (ATP)-dependent acylation of their cognate tRNA(s) with a specific amino acid. They can be seen as an index to reflect the energy metabolic rate of ischemic brain cells in ischemic penumbra. This study examined the relationship between arginyl-tRNA synthetase (ArgRS), one of the AARS, and cerebral ischemia in rats. The model of middle cerebral artery occlusion (MCAO) was established in rats. The expression levels of ArgRS protein and mRNA were detected in rat brain tissues at different time points following MCAO by Western blotting and RT-PCR, respectively. The results showed that the MCAO model was successfully established. Western blotting and RT-PCR analysis revealed that the ArgRS protein and mRNA were expressed in brain cells in both ischemic and normal penumbra tissues. The expression levels of ArgRS protein and mRNA peaked at 6 h after MCAO and decreased gradually. At 24 h, the expression levels of ArgRs protein and mRNA in ischemic penumbral tissues were lower than those in normal tissues. The expression levels of ArgRS mRNA and protein in ischemic penumbra varied with ischemic time, suggesting that the energy metabolism of brain cells in penumbra changed dynamically after ischemia to ensure the endogenous self-protection of the body. The brain oxygen supply should be improved as soon as possible, especially within 6-12 h after ischemia, so as to meet the demand for energy metabolism in ischemic penumbra and make sure the cell structure remains stable.

Expression and properties of arginyl-tRNA synthetase from jack bean (Canavalia ensiformis).

The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology. The thioredoxin component and the His-tag portion of the fused protein could be removed with thrombin, resulting in a homogeneous product retaining an N-terminal extension of 3.2 kDa compared to the native arginyl-tRNA synthetase. Both full-length fusion and thrombin-treated products proved to be active in aminoacylation, with similar kinetic parameters.

Two forms of human cytoplasmic arginyl-tRNA synthetase produced from two translation initiations by a single mRNA.

Human cytoplasmic arginyl-tRNA synthetase (ArgRS) is a component of a macromolecular complex consisting of at least nine tRNA synthetases and three auxiliary proteins. In mammalian cells, ArgRS is present as a free protein as well as a component of the complex. Via an alignment of ArgRSs from different vertebrates, the genes encoding full-length human cytoplasmic ArgRS and an N-terminal 72-amino acid deletion mutant (hcArgRS and DeltaNhcArgRS, respectively) were subcloned and expressed in Escherichia coli. The two ArgRS products were expressed as a soluble protein in E. coli. The level of production of DeltaNhcArgRS in E. coli and its specific activity were higher than those for hcArgRS. By Western blot analysis, using an antibody against the purified DeltaNhcArgRS, the two forms of ArgRS were detected in three human cell types. The 5'-end cDNA sequence, as confirmed by 5'RACE (5'-rapid amplification of cDNA ends), contained three start codons. Through mutation of the three codons, the two human cytoplasmic ArgRSs were found to be produced in different amounts, indicating that they resulted from two different translation initiation events. Here we show evidence that two forms of human cytoplasmic ArgRS were produced from two translational initiations by a single mRNA.

Biosynthesis and characterization of 4-fluorotryptophan-labeled Escherichia coli arginyl-tRNA synthetase.

Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher Km values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of 19F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.

Cloning and characterization of cDNA encoding a human arginyl-tRNA synthetase.

Arginyl-tRNA synthetase (ArgRS) plays a key role in protein synthesis as part of a multienzyme complex with a number of other aminoacyl-tRNA synthetase (aaRS) enzymes. We have isolated a full-length cDNA encoding ArgRS as part of a project on complementation of radiosensitivity in human cells with an Epstein-Barr Virus (EBV) vector-based human cDNA library. DNA sequence analysis identified an open reading frame of 1983 nucleotides with 87% homology to other mammalian ArgRS genes. The deduced amino acid (aa) sequence (661 aa) showed 87.7% identity to the Chinese hamster ovary (CHO) enzyme and 37.7% identity to the homologous Escherichia coli enzyme. Northern blot analysis revealed the presence of a single mRNA species of approx. 2.2 kb. The results described here demonstrate that ArgRS is highly conserved in mammalian cells and confirm the presence of a hydrophobic N-terminal region in the higher-molecular-weight complexed form of ArgRS.

A gene encoding arginyl-tRNA synthetase is located in the upstream region of the lysA gene in Brevibacterium lactofermentum: regulation of argS-lysA cluster expression by arginine.

The Brevibacterium lactofermentum argS gene, which encodes an arginyl-tRNA synthetase, was identified in the upstream region of the lysA gene. The cloned gene was sequenced; it encodes a 550-amino-acid protein with an M(r) of 59,797. The deduced amino acid sequence showed 28% identical and 49% similar residues when compared with the sequence of the Escherichia coli arginyl-tRNA synthetase. The B. lactofermentum enzyme showed the highly conserved motifs of class I aminoacyl-tRNA synthetases. Expression of the argS gene in B. lactofermentum and E. coli resulted in an increase in aminoacyl-tRNA synthetase activity, correlated with the presence in sodium dodecyl sulfate-polyacrylamide gels of a clear protein band that corresponds to this enzyme. One single transcript of about 3,000 nucleotides and corresponding to the B. lactofermentum argS-lysA operon was identified. The transcription of these genes is repressed by lysine and induced by arginine, showing an interesting pattern of biosynthetic interlock between the pathways of both amino acids in corynebacteria.

Transient rates of synthesis of five amionacyl-transfer ribonucleic acid synthetases during a shift-up of Escherichia coli.

The steady-state levels of a number of aminoacyl-transfer ribonucleic acid synthetases are known to be positively correlated with growth rate in Escherichia coli. To describe the regulation of these enzymes during a nutritional shift-up, use was made of the recent identification of polypeptide chains of several synthetases in whole cell lysates resolved by the O'Farrell two-dimensional gel system. A culture growing in acetate minimal medium was shifted to glucose-rich medium and pulse labeled with [3H]leucine and [3H]isoleucine for 30- or 6-s intervals during the 20 min after the shift. After mixing with a uniformly [35S]sulfate-labeled reference culture, the samples were subjected to two-dimensional gel electrophoresis. The 3H/35S ratio in the resolved synthetase polypeptides provided an accurate estimation of their transient rates of synthesis. Five aminoacyl-transfer ribonucleic acid synthetases (those for argnine, glycine, isoleucine, phenylalanine, and valine) exhibited an increase in formation within 30 to 90 s after the shift-up. The magnitude of the increases corresponded to the final steady-state values and were reached within 2 to 3 min. The addition to rifampin revealed that the increase in the differential rate of valyl-transfer ribonucleic acid synthetase formation was the result of increased messenger ribonucleic acid transcription and not of a relaxation of some translation restriction.