Human alanine-glyoxylate aminotransferase 2 lowers asymmetric dimethylarginine and protects from inhibition of nitric oxide production

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.

FIGURE 1.

Expression and localization of human AGXT2 in COS-7 cells. A, immunoblotting of COS-7 cells infected with AdGFP (Control Vector) or AdAGXT2FLAG. Cell lysates were probed with anti-FLAG (upper panel) or anti-β-actin (lower panel) antibodies. B, intracellular localization of human AGXT2FLAG in COS-7 cells. COS-7 cells were infected with either the AdCre control vector or AdAGXT2FLAG, stained with MitoTracker and anti-FLAG antibodies. Fluorescence confocal microscopy was performed to detect GFP (green), MitoTracker (red), FLAG (blue), or a merged image of all three markers (Merge).

FIGURE 2.

Intracellular localization of human AGXT2 in HUVEC. HUVEC were infected with AdAGXT2FLAG and stained with anti-FLAG antibodies and either: A, MitoTracker; B, anti-GRP78 (a marker of endoplasmic reticulum); C, anti-giantin (a marker of the Golgi); or D, anti-PMP70 (a marker of peroxisomes). Fluorescence confocal microscopy was performed to detect GFP (green), MitoTracker (red), GRP78 (red), giantin (red), PMP70 (red), FLAG (blue), or a merged image of multiple markers (Merge).

FIGURE 3.

Determination of the mitochondrial cleavage sequence of human AGXT2. A, human AGXT2FLAG was purified from a mitochondrial preparation of AdAGXT2FLAG-infected COS-7 cells by FLAG affinity chromatography, separated by SDS-PAGE, transferred to PVDF membranes, and stained with Coomassie Blue to identify a major mature protein of 52 kDa and a minor precursor protein of 60 kDa. The 52-kDa band was excised from the membrane and subjected to partial N-terminal sequencing. B, N-terminal sequencing results aligned with the predicted N-terminal amino acid sequence of full-length human AGXT2.

FIGURE 4.

Expression of human AGXT2 in mice. A, C57BL/6 mice were injected retro-orbitally with AdGFP (lanes 1–3), AdAGXT2FLAG (lanes 4–6), or AdDDAH1 (lanes 7–9). Four days after injection, the livers were harvested and subjected to immunoblotting with antibodies to DDAH1, FLAG, or β-actin. Three separate mice were injected with each adenovirus. B, total AGXT activity of liver lysates prepared 4 days after injection of AdEmpty (n = 4), AdAGXT2FLAG (n = 4), or AdDDAH1 (n = 4). C, levels of ADMA in the liver and plasma 4 days after injection of AdEmpty (n = 10 for liver and n = 25 for plasma), AdAGXT2FLAG (n = 10 for liver and n = 25 for plasma), or AdDDAH1 (n = 5 for liver and n = 5 for plasma). Values are mean ± S.E. *, p < 0.05 compared with mice infected with AdEmpty.

FIGURE 5.

Effect of AGXT2 on production in endothelial cells. A, immunoblotting of lysates of MS-1 cells infected with AdGFP, AdAGXT2FLAG, or AdDDAH1. Membranes were probed with antibodies to DDAH1 (Anti-DDAH1), FLAG (Anti-FLAG), or β-actin (Anti-β-actin). B, total nitrite levels in the conditioned medium from MS-1 cells infected with AdEmpty, AdAGXT2FLAG, or AdDDAH1 and incubated with or without 100 μm ADMA for 24 h (n = 3, representative of five separate experiments). Values are mean ± S.E. *, p < 0.05 compared with cells infected with AdEmpty.