Abstract
Elevated blood concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric-oxide (NO) synthase, are found in association with diabetes, hypertension, congestive heart failure, and atherosclerosis. ADMA levels are controlled by dimethylarginine dimethylaminohydrolases (DDAHs), cytosolic enzymes that hydrolyze ADMA to citrulline and dimethylamine. ADMA also has been proposed to be regulated through an alternative pathway by alanine-glyoxylate aminotransferase 2 (AGXT2), a mitochondrial aminotransferase expressed primarily in the kidney. The goal of this study was to define the subcellular localization of human AGXT2 and test the hypothesis that overexpression of human AGXT2 protects from ADMA-induced inhibition in nitric oxide (NO) production. AGXT2 was cloned from human kidney cDNA and overexpressed in COS-7 cells and human umbilical vein endothelial cells with a C-terminal FLAG epitope tag. Mitochondrial localization of human AGXT2 was demonstrated by confocal microscopy and a 41-amino acid N-terminal mitochondrial cleavage sequence was delineated by N-terminal sequencing of the mature protein. Overexpression of human AGXT2 in the liver of C57BL/6 mice using an adenoviral expression vector produced significant decreases in ADMA levels in plasma and liver. Overexpression of human AGXT2 also protected endothelial cells from ADMA-mediated inhibition of NO production. We conclude that mitochondrially localized human AGXT2 is able to effectively metabolize ADMA in vivo resulting in decreased ADMA levels and improved endothelial NO production.
Highlights
Vated blood levels of ADMA are associated with increased cardiovascular morbidity and mortality, suggesting that ADMA may be an independent cardiovascular risk factor [2]
Our results demonstrate that human AGXT2 is localized in mitochondria and contains a 41-amino acid N-terminal mitochondrial cleavage sequence
Despite widespread interest in ADMA as a cardiovascular risk factor and a large amount of recent work on the regulation of ADMA by dimethylarginine dimethylaminohydrolases (DDAHs) [1, 31], relatively little is known about the role of human AGXT2 in ADMA metabolism
Summary
Vated blood levels of ADMA are associated with increased cardiovascular morbidity and mortality, suggesting that ADMA may be an independent cardiovascular risk factor [2]. AGXT2, but not AGXT1, can utilize ADMA as an amino donor, leading to the formation of DMGV [17] This pathway of ADMA metabolism is likely to occur in vivo, because ADMA-derived DMGV and a related metabolite, ␣-keto-␦-(N,N-dimethylguanidino)butyric acid (DMGB) have been observed to accumulate in the urine after rats are injected with radiolabeled ADMA [18]. Mutations that result in the mistargeting of human AGXT1 from peroxisomes to mitochondria can cause primary hyperoxaluria type 1 [19], an autosomal recessive disorder of oxalate metabolism [20, 21] This observation suggests that altered subcellular localization of AGXT1 can significantly affect its function. The intracellular localization of human AGXT2 has not been reported, and it is not known whether mitochondrial AGXT2 can metabolize ADMA in vivo
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