High Maternal Glycine Levels Increase the Risk of Developing Atrial Septal Defect in the Offspring

Abstract

Amino acid imbalance is linked to increased congenital heart disease risk. Here, we found women carrying rs2545801 C/C genotypes exhibited increased glycine levels and increased risk for atrial septal defects (ASDs) in their offspring. Elevated maternal glycine levels during the first trimester were correlated with a higher ASD risk in the offspring. Additionally, feeding pregnant mice with high-glycine chow increased ASD risk in their offspring. Mechanistically, elevated maternal glycine led to increased lysine-glycylation of lysine-688 within the TEK receptor tyrosine kinase and inhibited TEK-PI3K-AKT/FOXO1 signaling in cardiac endothelial cells. These findings indicate that lysine-glycylation exerts teratogenic effects and may be a target for ASD intervention.

Keywords: TEK; atrial septal defects; glycine; maternal single-nucleotide variation; pregnancy serum.

Graphical abstract

Figure 1

Increased Maternal Glycine Is Associated With the Risk for Developing Atrial Septal Defect in Embryos (A) Serum arginine and glycine concentrations in individuals with T/T (n = 9), T/C (n = 7), and C/C (n = 8) genotypes; C/C genotype shows higher glycine levels. (B) Plasma glycine concentration in pregnant women at 10 to 12 weeks gestation with normal or offspring with congenital heart disease. n = 97 for control group, n = 17 for the atrial septal defect (ASD) group, n = 45 for the ventricular septal defect (VSD) group, n = 11 for tetralogy of Fallot (TOF) group, n = 9 for transposition of the great arteries (TGA) group. (C) ASD phenotype in embryos from mice with high levels of glycine. Representative histological staining results are shown (scale bar, 500 μm). (D) Number of healthy and embryos with atrial septal defect from pregnant mice fed with normal chow or 10%-glycine chow (n = 12 mice each group). (E) Percentage of offspring with atrial septal defect from pregnant mice fed with normal chow or 10%-glycine chow (n = 12 mice each group). (F) Glycine concentrations in embryonic heart tissues increased in 10%-glycine chow group (n = 12 mice each group). Data are presented as mean ± SD. Statistical analysis among 2 groups was performed using a 2-tailed unpaired Student’s t-test. ^nsP > 0.05, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 2

TEK Is Glycylated at Lysine 688 (A) Tandem affinity purification assay identified 302 proteins as potential glycyl-tRNA synthetase (GARS)-interacting proteins, and 217 proteins were identified with K-Gly modification in a tryptic peptide library. TEK represents the exclusive intersection of the 2 sets. (B) Lysine 688 site of TEK, identified as being glycylated. MS/MS spectra result of tryptic peptides from human umbilical vein endothelial cells (HUVECs) (top), with the synthetic glycylated peptide bearing the same peptide sequence (bottom). (C) GARS bound to TEK. GARS and TEK interactions are examined by coimmunoprecipitation against exogenous Flag-tagged TEK (left) and Flag-tagged GARS (right). (E) Endogenous GARS interacts with TEK in human atrial tissue. Immunoprecipitation against TEK was performed before Western blot analysis. (F) MS/MS spectra of TEK K688Gly-containing peptides from mice heart tissue.

Figure 3

GARS Interacts and Catalyzes Lysine Glycylation of TEK (A) Purified glycyl-tRNA synthetase (GARS) and the proven enzyme inactive mutants GARS^G580R and GARS^H472R are assessed for their ability to catalyze lysine glycylation (K-Gly) formation in a synthetic K688 peptide in vitro. (B and C) Glycine increases K-Gly levels of endogenous (B) and exogenously-expressed (C) TEK in a dose-dependent manner in HUVEC. (Right) Quantification of relative intensity of K-Gly blots. n = 3 biological replicates. (D and E) GARS, but not the enzyme inactive mutants GARS^G580R and GARS^H472R, increases the K-Gly modification of endogenous (D) and exogenously expressed (E) TEK in HUVEC. (Right) Quantification of relative intensity of K-Gly blots. n = 3 biological replicates. (F and G) GARS knockdown deceases K-Gly levels of endogenous (F) and exogenously expressed (G) TEK in HUVEC. (Right) Quantification of relative intensity of K-Gly blots. n = 3 biological replicates. (H) Glycine increases K-Gly levels of wild-type but not K688R mutant TEK. (Right) Quantification of relative intensity of K-Gly blots. n = 3 biological replicates. (I) GARS overexpression increases K-Gly level of wild-type but not K688R mutant TEK. Immunoprecipitation against Flag was performed before Western blot analysis. (Right) Quantification of relative intensity of K-Gly blots. n = 3 biological replicates. Data are presented as mean ± SD. Statistical analysis among 2 groups was performed using a 2-tailed unpaired Student’s t-test. ^nsP > 0.05, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Abbreviations as in Figure 2.

Figure 4

Lysine Glycylation Impedes TEK Membrane Localization and Inactivates TEK (A and B) Glycine does not affect the protein (A) and mRNA (B) levels of TEK in HUVEC. n = 5 biological replicates. P values are calculated using the 2-tailed Student's t-test. (C) The TEK K688 site is evolutionarily conserved across species. (D and E) TEK membrane localization in cultured cells subjected to various treatments as examined via immunostaining (D) and Western blot (E). Scale bar represents 10 μm. (Right) Quantification of relative intensity of Flag. n = 3 biological replicates. (F and G) Intracellular localization of the TEK^K688G mutant, which mimics the glycylation status, examined via Western blot (F) and immunostaining (G). Scale bar represents 10 μm. (Right) Quantification of relative intensity of Flag. n = 3 biological replicates. (H) The phosphorylation levels of S473 of AKT and S256 of FOXO1 and the protein levels of ANG2 in HUVEC subjected to glycine treatment or not. (I) The nuclear localization of FOXO1 in HUVEC subjected to glycine treatment or not. (Right) Quantification of relative intensity of FOXO1. n = 3 biological replicates. (J) The mRNA levels of FOXO1 transcriptional activity targets in HUVEC subjected to different treatments. n = 5 biological replicates. P values are calculated using the 2-tailed Student's t-test. (K and L) Intracellular localization of TEK and TEK^K688R mutant, which impeded the lysine glycylation, examined via Western blot (K) and immunostaining (L). Scale bar represents 10 μm. (Right) Quantification of relative intensity of Flag. n = 3 biological replicates. (M) The mRNA levels of FOXO1 transcriptional activity targets in HUVEC subjected to different treatments; n = 5 biological replicates. P values are calculated using the two-tailed Student's t-test. (N) The phosphorylation levels of S473 of AKT and S256 of FOXO1 and the protein levels of ANG2 in HUVEC subjected to different treatments. (Right) Quantification of relative intensity of ANG2. n = 3 biological replicates. Data are presented as mean ± SD. Statistical analysis among 2 groups was performed using a 2-tailed unpaired Student’s t-test. ^nsP > 0.05, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 5

SIRT3 Deglycylates and Activates TEK (A) SIRT3, but not other sirtuins, decreases K-Gly levels of ectopically-expressed TEK. (Right) Quantification of relative intensity of K-Gly. n = 3 biological replicates. (B) SIRT3 and TEK interactions are examined via coimmunoprecipitation in HUVECs. (C and D) SIRT3 catalyzes deglycylation reactions in vitro (C), in contrast to the deaminoacylase-defective mutant SIRT3^H248A (D). (E to G) SIRT3 decreases the K-Gly levels of total protein (E) and exogenous (F) and endogenous TEK (G) in HUVEC. (Right) Quantification of relative intensity of ANG2. n = 3 biological replicates. (H and I) TEK membrane localization in cultured cells overexpressed SIRT3 or SIRT3^H248A, examined via immunostaining (H) and Western blot (I); the scale bar represented 10 μm. (Bottom) Quantification of relative intensity of TEK. n = 3 biological replicates. (J) The protein levels of ANG2, the phosphorylation levels of S473 of AKT and S256 of FOXO1 in HUVEC subjected to SIRT3 or SIRT3^H248A. (Right) Quantification of relative intensity of ANG2. n = 3 biological replicates. (K) The mRNA levels of FOXO1 transcriptional activity targets in HUVEC subjected to SIRT3 or SIRT3^H248A; n = 5 biological replicates. (L and M) SIRT3 knockdown increases K-Gly levels of total protein (L) and TEK (M) in HUVEC. (Bottom) Quantification of relative intensity of K-Gly. n = 3 biological replicates. (N and O) Intracellular localization of the TEK (N) and FOXO1 (O) in HUVEC subjected to different treatments, examined via Western blot. (Bottom) Quantification of relative intensity of TEK (N) and FOXO1 (O). n = 3 biological replicates. (P) SIRT3 knockdown increases the protein levels of ANG2, reduces the phosphorylation levels of S473 of AKT and S256 of FOXO1 in HUVEC. (Right) Quantification of relative intensity of ANG2. n = 3 biological replicates. (Q) The mRNA levels of FOXO1 transcriptional activity targets in HUVEC subjected to SIRT3 knockdown; n = 5 biological replicates. Data are presented as mean ± SD. Statistical analysis among 2 groups was performed using a 2-tailed unpaired Student’s t-test. ^nsP > 0.05, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Abbreviations as in Figure 2.