Gene structure, localization and role in oxidative stress of methionine sulfoxide reductase A (MSRA) in the monkey retina

Abstract

MSRA (EC 1.8.4.6) is a member of the methionine sulfoxide reductase family that can reduce methionine sulfoxide (MetO) in proteins. This repair function has been shown to protect cells against oxidative damage. In this study we have assembled the complete gene structure of msrA and identified the presence of two distinct putative promoters that generate three different transcripts. These transcripts were cloned by 5'RACE and code for three MSRA isoforms with different N-termini. The different forms of MSRA target to distinct intracellular regions. The main MSRA transcript (msrA1) had been previously shown to target the mitochondria. MsrA2 and 3 originate from a second promoter and target the cytosol and nuclei. In the monkey retina msrA message was detected mainly in the macular RPE-choroid region while its activity was measured mainly in the soluble fractions of fractionated neural retina and RPE-choroid. The MSRA protein is found throughout the retina but is especially abundant at the photoreceptor synapses, ganglion and Müller cells. Interestingly, MSRA was not detected in the mitochondria of the photoreceptor inner segments. The RPE in the peripheral retina shows very low levels of expression but the RPE in the macular region is strongly labeled. Targeted silencing of msrA message rendered cultured RPE cells more sensitive to oxidative damage suggesting a role for MSRA in RPE protection against oxidative stress. Collectively these data suggest MSRA may play an important role in protecting macular RPE from oxidative damage.

Silencing of the methionine sulfoxide reductase A gene results in loss of mitochondrial membrane potential and increased ROS production in human lens cells

“…Although this possibility can not be ruled out, we do not believe that this condition contributes to the present data since similar effects have been observed upon deletion of MsrA in retinal cells which are not found in an anoxic environment (Lee et al, 2006). Moreover, no changes in ROS levels or mitochondrial membrane potential were seen in the mock transfected controls.…”

Protection of Mouse Embryonic Stem Cells from Oxidative Stress by Methionine Sulfoxide Reductases

“…The hybridization signal is low, and becomes evident on the X-ray film only after the longer form MsrA bands are overexposed ( Figure 10A, thin arrow). There are also protein bands with molecular weights of ~46 and ~48 kD indicating homodimers of MsrA long form proteins ( Figure 10A, arrow head), even with protein samples thoroughly treated by reducing agents before SDS-PAGE, as has been reported by other laboratories [Lee et al, 2006]. On our films, there is also a band with a molecular weight of 39 kD, possibly heterodimers formed by a long form protein of MsrA (MW: 23 kD) and a smaller form of MsrA protein (MW 16 kD) ( Figure 10A, *).…”

“…Currently, most studies on MsrA isoforms emphasize the 5' terminus where different isoforms for mitochondrial signal peptide are present and dictates where protein products are localized within the mitochondria [Kim & Gladyshev, 2006;Lee et al, 2006;Haenold et al, 2007]. 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, however, no detailed studies on these transcripts have been reported [Kim & Gladyshev, 2006].…”

“…MsrA1 transcript forms the longest protein that targets the mitochondria. MsrA2 and MsrB3 are formed from a second promoter and localize in the cytosol and nuclei [Lee et al, 2006;Pascual et al, 2009]. Two novel splice forms: MsrA2a and MsrA2b were found recently in rat smooth muscle cells [Haenold et al, 2007].…”

“…In addition, a minor band with a molecular weight of ~32 kD, possibly formed by homodimers of two molecules of the smaller form of MsrA proteins ( Figure 10A, ** ). The 32kD and 39kD bands cannot readily be explained by the dimerization from cytosolic isoforms of MsrA which are 19-20kD in size [Kim & Gladyshev, 2006;Lee et al, 2006]. To confirm the existence of this smaller form of protein, we have carried out RT-PCR using total RNA extracted from mouse embryonic stem cells to amplify the full cDNA [Jia et al, 2011].…”