Abstract
Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common AGXT2 single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as β-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding AGXT2 SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the AGXT2 SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p<0.001). ADMA concentrations were not affected by both SNPs. A haplotype analysis revealed that the second investigated AGXT2 SNP rs16899974, which was not significantly linked to the other AGXT2 SNP, further aggravates the effect of rs37369 with respect to BAIB concentrations in plasma and urine. To investigate the impact of the amino acid exchange p.Val140Ile, we established human embryonic kidney cell lines stably overexpressing wild-type or mutant (p.Val140Ile) AGXT2 protein and assessed enzyme activity using BAIB and stable-isotope labeled [2H6]-SDMA as substrate. In vitro, the amino acid exchange of the mutant protein resulted in a significantly lower enzyme activity compared to wild-type AGXT2 (p<0.05). In silico modeling of the SNPs indicated reduced enzyme stability and substrate binding. In conclusion, SNPs of AGXT2 affect plasma as well as urinary BAIB and SDMA concentrations linking methylarginine metabolism to the common genetic trait of hyper-β-aminoisobutyric aciduria.
Highlights
In various clinical studies elevated plasma concentrations of endogenously formed symmetric (SDMA) and asymmetric (ADMA) dimethylarginine were identified as prospective and independent risk markers of cardiovascular diseases and mortality [1,2,3]
It was shown that both dimethylarginines compete with L-arginine for uptake into the cell by cationic amino acid transporter 1 (CAT1) [6] and that ADMA acts as an endogenous inhibitor of nitric oxide synthases [7,8]
The AGXT2 single nucleotide polymorphisms (SNPs) rs37369 was associated with significantly higher plasma SDMA as well as plasma and urinary BAIB concentrations
Summary
In various clinical studies elevated plasma concentrations of endogenously formed symmetric (SDMA) and asymmetric (ADMA) dimethylarginine were identified as prospective and independent risk markers of cardiovascular diseases and mortality [1,2,3]. Elevation of methylarginine concentrations may indicate structural or functional deficiencies of the metabolizing enzyme(s) dimethylarginine dimethylaminohydrolase (DDAH1 and DDAH2, which degrade ADMA) and alanine-glyoxylate aminotransferase 2 (AGXT2, which degrades ADMA and SDMA). These enzymes have further substrates (as detailed below) [10] and may be involved in alternative regulatory mechanisms [11]. A recent study linked SDMA plasma concentrations in humans with polymorphisms within the AGXT2 gene [13]. The in vivo studies were complemented by in silico studies regarding both SNPs as well as in vitro studies for the AGXT2 SNP rs37369
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