Alanine-glyoxylate aminotransferase 2 (AGXT2) polymorphisms have considerable impact on methylarginine and β-aminoisobutyrate metabolism in healthy volunteers - PubMed (original) (raw)

Alanine-glyoxylate aminotransferase 2 (AGXT2) polymorphisms have considerable impact on methylarginine and β-aminoisobutyrate metabolism in healthy volunteers

Anja Kittel et al. PLoS One. 2014.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. AGXT2 SNPs and biochemical measures.

The AGXT2 SNP rs37369 was associated with significant differences in plasma SDMA (A), plasma (E) and urinary BAIB (G) concentrations, whereas AGXT2 SNP rs16899974 was associated with significant differences in plasma (F) and urinary (H) BAIB concentrations. Plasma SDMA concentrations were not significantly different in case of rs16899974 (B), as well as urinary SDMA concentrations for both SNPs (C–D). Values are median ±1.5 IQR. Details on the underlying genotype distribution are shown in table 2 and in table S1. The Jonckheere-Terpstra trend-test (p<0.001 in all cases except for figure 1B–D) and Kruskal-Wallis test (Dunn’s post-test, * p<0.05, ** p<0.01, *** p<0.001) were used for statistical analysis. ADMA, asymmetric dimethylarginine; AGXT2, alanine-glyoxylate aminotransferase 2; BAIB, β-aminoisobutyrate; Hetero, heterozygous for minor allele; Homo, homozygous for minor allele; SDMA, symmetric dimethylarginine; WT (wild-type), homozygous for major allele.

Figure 2

Figure 2. Characterization of HEK cell lines overexpressing human wild-type and mutant (rs37369, p.Val140Ile) AGXT2 protein.

HEK cells overexpressing human wild-type (AGXT2 WT) and mutant (AGXT2 rs37369) protein showed similar AGXT2 protein expression levels determined by immunoblot (A, representative immunoblot; 20 µg total protein per line) and immunofluorescence (B) analysis. AGXT2 was localized to mitochondria in both cell lines. Cells transfected with the empty vector (VC) were used as negative control. Enzyme activity of mutant AGXT2 indicated significant reduction of [2H6]-SDMA degradation (C; each n = 10), [2H6]-DM’GV formation (D; each n = 10) and BAIB degradation (E; each n = 5) compared to wild-type protein normalized to total protein. Area ratio is defined as [2H6]-DM’GV/DM’GV. Values are median ±1.5 IQR. Unpaired 2-tailed Student’s t test (* p<0.05, *** p<0.001) was used for statistical analysis. AGXT2, alanine-glyoxylate aminotransferase 2; DM’GV, (N,N’-dimethyl-guanidino)valeric acid (AGXT2-dependent metabolite of SDMA); HEK cells, human embryonic kidney 293 cells; VC, vector control; WT, wild-type.

Figure 3

Figure 3. Prediction of the phenotypic effects of the coding SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu) in AGXT2 using structural information from in silico homology modeling.

(A) Three-dimensional model of the AGXT2 (V140I) dimer showing the subunits A (blue) and B (cyan) in space-filled presentation. Residues Q83 (chain A), I140 (chain B), and V498 (chain A) are colored in orange, yellow and magenta, respectively. The β-aminoisobutyrate substrate of subunit A is colored according to the atom types. This view shows that V498 is buried in the interior of the molecule, whereas I140 of chain B is located close to the substrate binding site of subunit A. (B) Enlargement of the site of the V140I mutation. Residues 60–120 of chain A (blue) and residues 120–150 of chain B (cyan) are shown in ribbon presentation. A red arrow denotes a clash between the side chains of I140 (chain B; yellow) and Q83 (chain A; orange) that is not observed for V140 in the wild-type. (C) Enlargement of the C-terminal residues 400–500 showing the effect of a V498L mutation. The larger side chain of L498 forms steric clashes with the methylene groups of the K417 side chain (denoted by a red arrow).

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