Identification of a new functional splice variant of the enzyme methionine sulphoxide reductase A (MSRA) expressed in rat vascular smooth muscle cells

Abstract

Reactive oxygen species contribute to ageing of the vascular system and development of cardiovascular disease. Methionine-S-sulphoxide, an oxidized form of methionine, is repaired by the enzyme methionine sulphoxide reductase A (MSRA). The enzyme, targeted to mitochondria or the cytosol by alternative splicing, is vital for oxidative stress resistance. This study was designed to examine the endogenous expression and intracellular localization of MSRA in rat aortic vascular smooth muscle cells (VSMCs). We detected robust MSRA immunoreactivity exclusively in mitochondria. Sequence analysis of msrA transcripts revealed the presence of a novel mitochondrial splice variant, msrA2a, in cultured rat VSMCs as well as in aortic tissue preparations. The enzymatic activity of a recombinant MSRA2a protein was confirmed by the reduction of methionine sulphoxide in a model substrate peptide. We conclude that multiple MSRA variants participate in the repair of oxidized proteins in VSMC mitochondria, but that other protective mechanisms may exist in the cytoplasmic compartment.

Over-expression of methionine sulfoxide reductase A in the endoplasmic reticulum increases resistance to oxidative and ER stresses

“…The most abundant MsrA form has a typical N-terminal mitochondrial targeting sequence [7], but is distributed in the mitochondria, cytosol, and nucleus [8][9][10]. Other known MsrA variants are targeted to either the cytosol and nucleus or the mitochondria [5,6,11]. Interestingly, there is no evidence for the localization of MsrA in the ER or the presence of an ER-targeted MsrA form.…”

“…MsrB1 (a selenoprotein) is present in the cytosol and nucleus, MsrB2 localized in the mitochondria, and MsrB3 is targeted to the endoplasmic reticulum (ER). In contrast, a single MsrA gene exists in mammals [4], although multiple MsrA variants generated by alternative splicing have been reported in mammalian cells [5,6]. The most abundant MsrA form has a typical N-terminal mitochondrial targeting sequence [7], but is distributed in the mitochondria, cytosol, and nucleus [8][9][10].…”

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

“…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].…”

“…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]. Alternative splicing occurred in the second exon with the MsrA2, a functional isoform.…”

Expression, subcellular localization, and antioxidant role of mammalian methionine sulfoxide reductases in Saccharomyces cerevisiae

“…Mammalian genomes encode a single MsrA gene, but alternatively spliced forms have also been identified (4)(5)(6). The most abundant form of MsrA harbors a typical mitochondrial signal peptide at its N-terminus, but interestingly this MsrA protein is detected in both the cytosol and mitochondria (7,8).…”