Extracellular glycine is necessary for optimal hemoglobinization of erythroid cells

Abstract

Vertebrate heme synthesis requires three substrates: succinyl-CoA, which regenerates in the tricarboxylic acid cycle, iron and glycine. For each heme molecule synthesized, one atom of iron and eight molecules of glycine are needed. Inadequate delivery of iron to immature erythroid cells leads to a decreased production of heme, but virtually nothing is known about the consequence of an insufficient supply of extracellular glycine on the process of hemoglobinization. To address this issue, we exploited mice in which the gene encoding glycine transporter 1 (GlyT1) was disrupted. Primary erythroid cells isolated from fetal livers of GlyT1 knockout (GlyT1^-/-) and GlyT1-haplodeficient (GlyT1^+/-) embryos had decreased cellular uptake of [2^-14C]glycine and heme synthesis as revealed by a considerable decrease in [2-¹⁴C]glycine and ⁵⁹Fe incorporation into heme. Since GlyT1^-/- mice die during the first postnatal day, we analyzed blood parameters of newborn pups and found that GlyT1^-/- animals develop hypochromic microcytic anemia. Our finding that Glyt1-deficiency causes decreased heme synthesis in erythroblasts is unexpected, since glycine is a non-essential amino acid. It also suggests that GlyT1 represents a limiting step in heme and, consequently, hemoglobin production.

Figure 1.

GlyT1 expression increases during erythroid differentiation in vitro and in vivo. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of (A) GlyT1 and (B) globin mRNA expression in primary erythroid cells kept either in a non-differentiating state (0 h) or induced to differentiate for 24 to 72 h in vitro. (C) Western blot analysis of GlyT1 protein expression in primary erythroid cells differentiated for 0 h, 24 h and 48 h. (D) qRT-PCR and (E) western blot analysis of GlyT1 expression in Ter119⁻ and Ter119⁺ cells isolated from mouse bone marrow. qRT-PCR-based results are presented as fold-change relative to the 0 h sample. Statistical analysis was done using one way analysis of variance followed by a Bonferroni multiple comparison test (n=3). The P-value refers always to the value of the previous time point; *P<0.05; **P<0.01; ***P<0.001.

Figure 2.

[2-¹⁴C]glycine uptake and incorporation into heme is disrupted in GlyT1-depleted erythroid cells. Erythroid cells isolated from GlyT1^+/+, GlyT1^+/− and GlyT^−/−embryos were either kept in a non-differentiating state (0 h) or induced to differentiate for 24 and 48 h. (A) Giemsa staining of GlyT1^+/+, GlyT1^+/− and GlyT^−/− undifferentiated (0 h) and differentiated (24 h and 48 h) fetal liver cells (B) Total uptake of [2-¹⁴C]glycine and (C) [2-¹⁴C]glycine incorporation into heme. [2-¹⁴C]glycine values are presented relative to the wild-type cells at indicated time intervals. Statistical analysis was done using one way analysis of variance followed by a Bonferroni multiple comparison test (n=3). The P value always refers to the value of the previous time point; *P<0.05; **P<0.01; ***P<0.001.

Figure 3.

⁵⁹Fe uptake from ⁵⁹Fe-Tf and ⁵⁹Fe incorporation into heme, as well as hemoglobin levels, are significantly reduced in GlyT1^−/− erythroid cells. Erythroid cells isolated from GlyT1^+/+, GlyT1^+/− and GlyT^−/− embryos were either kept in a non-differentiating state (0 h) or induced to differentiate for 24 and 48 h. (A) Total uptake of ⁵⁹Fe from ⁵⁹Fe-Tf, (B) ⁵⁹Fe incorporation into heme and (C) hemoglobin levels. ⁵⁹Fe and hemoglobin values are presented relative to the wild-type cells at indicated time intervals. Statistical analysis was done using one way analysis of variance followed by a Bonferroni multiple comparison test (n=3); *P<0.05; **P<0.01; ***P<0.001.

Figure 4.

Cell surface transferrin receptor levels are reduced in differentiating erythroid cells depleted of GlyT1. Erythroid cells were isolated from GlyT1^+/+, GlyT1^+/− and GlyT1^−/− embryos and either kept in a non-differentiated state (0 h) or induced to differentiate for 24 to 48 h. TfR surface expression was analyzed by flow cytometry. (A) Representative TfR (CD71) fluorescence histograms obtained from cells of each genotype at indicated time points. (B) TfR quantification based on mean fluoresce intensity displayed as values relative to those in 0 h wild-type cells. Statistical analysis was done using one way analysis of variance followed by a Dunnett multiple comparison test in relation to the respective GlyT^+/+ sample (n=6); *P<0.05; **P<0.01;***P<0.001.

Figure 5.

GlyT1^−/− embryos are paler than their wild-type and heterozygous counterparts. Embryos were isolated at embryonic days 12.5 (E12.5), 14.5 (E14.5) and 16.5 (E16.5). Genotype was determined by polymerase chain reaction analysis as described in the Methods section.