8 research outputs found
Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia
Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to
synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the
mitochondria of red blood cell precursors. A common subtype of congenital sideroblastic
anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment
for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled
with iron chelation. The function of SLC25A38 is not known. Here we report that the
SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters
required for the initiation of heme synthesis. To do so, we took advantage of the fact that
mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis
of folate derivatives through the glycine cleavage system. The data were consistent with
Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25
family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on
these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid
(5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to
restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine
nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish
model, we determined that the addition of folate with glycine was able to restore hemoglobin
levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in
zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability
of glycine and folate in humans, this study points to a potential novel treatment for
SLC25A38 congenital sideroblastic anemia.Genome Canada as large-scale applied research project with funding contributions from the Dalhousie Medical Research Foundation, the Nova Scotia Research Innovation Trust, and the Nova Scotia Department of Health and Wellnes
Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia
AbstractSideroblastic anemias are acquired or inherited anemias that result in a decreased ability tosynthesize hemoglobin in red blood cells and result in the presence of iron deposits in themitochondria of red blood cell precursors. A common subtype of congenital sideroblasticanemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatmentfor SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupledwith iron chelation. The function of SLC25A38 is not known. Here we report that theSLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transportersrequired for the initiation of heme synthesis. To do so, we took advantage of the fact thatmitochondrial glycine has several roles beyond the synthesis of heme, including the synthesisof folate derivatives through the glycine cleavage system. The data were consistent withHem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25family member Ymc1, as a potential secondary mitochondrial glycine importer. Based onthese findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid(5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able torestore heme levels to normal in yeast cells lacking Hem25 function. While neither glycinenor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafishmodel, we determined that the addition of folate with glycine was able to restore hemoglobinlevels. This difference is likely due to the fact that yeast can synthesize folate, whereas inzebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerabilityof glycine and folate in humans, this study points to a potential novel treatment forSLC25A38 congenital sideroblastic anemia
Cytosolic threonine aldolase is the main soruce of glycine for heme synthesis.
<p>A) Glycine can be synthesized in glucose grown yeast via three enzymes, Gly1 (threonine aldolase), and cytosolic (Shm2) and mitochondrial (Shm1) serine hydroxymethyltransferases. B) Yeast cells of the indicated genotypes were grown to mid-log phase and cells were processed for glycine and heme determination. Wild type glycine was 6.0 nmol/10<sup>8</sup> cells. C) Cells of the indicated genotypes were grown to mid-log phase and 1:10 serial dilutions plated on SD medium with no supplements.</p
Identification of a putative second mitochondrial glycine importer.
<p>Yeast cells of the indicated genotypes were grown to mid-log phase in the presence of no supplements or the addition of 5 mM glycine or 50 ÎĽg/ml 5-Ala. Cells were processed for heme determination.</p
Rescue of the zebrafish model of congenital sideroblastic anemia.
<p>A) Whole-mount <i>in situ</i> hybridizations with probes for <i>slc25a38a</i> and <i>slc25a38b</i> at 24 and 34 hpf stages of development demonstrate predominant erythroid expression. For each stage and probe, head and tail views are shown and sites of expression are labeled. Abbreviations: pbi–posterior blood island, pcv–posterior cardinal vein, ss–somites, bl–blood, ret–retina, br–brain, h–heart. Expression of <i>slc25a38b</i> was observed in the posterior blood island, blood and posterior cardinal vein consistent with its preferential expression in erythroid progenitors and erythrocytes, whereas <i>slc25a38a</i> had the same blood-related expression pattern features and was also expressed in retina, brain and somites at 24 hpf. B) Representative images for hemoglobin staining using <i>o</i>-dianisidine of 48 hpf zebrafish embryos injected with <i>slc25a38a+b</i> morpholinos, or standard control morpholino (STD MO), treated with 100 mM glycine, 1 mM sodium folate, or both, starting from 4 hours post-injection. To control for morpholino specificity, we also injected 5-mismatch (5MM) versions of the same <i>slc25a38a+b</i> morpholinos and stained the injected embryos with o-dianisidine. For STD MO and 5MM morphants, images from only the STD MO untreated group are presented because of the low variation in <i>o</i>-dianisidine scores of untreated embryos versus those where glycine and/or folate were present. C) Graph of the <i>o</i>-dianisidine staining heme scores of 48 hpf zebrafish embryos injected with either <i>slc25a38a+b</i> morpholinos (slcMO) or STD MO and then treated from 4 through to 48 hpf with glycine, folate, or both glycine plus folate. All embryos were scored as having “low”, “medium” or “normal” hemoglobin levels in a blinded manner based on visual inspection of <i>o</i>-dianisidine staining. The total numbers (right axis) of scored embryos are indicated, from a total of three independent experiments. The “***” indicates that the <i>p</i>-value between the numbers of embryos in different scoring categories for <i>slc25a38a+b</i> morphants treated with glycine and folate versus the untreated <i>slc25a38a+b</i> morphant group is <0.001.</p
Hem25 is required for the effective import of glycine into mitochondria.
<p>A) Glycine can serve as the sole nitrogen source in <i>S</i>. <i>cerevisiae</i>. The GCV converts glycine to NH<sub>3</sub>, as the GCV resides in the mitochondria the use of glycine as a nitrogen source requires efficient uptake of glycine into the mitochondria. B) Inactivation of the <i>HEM25</i> gene in yeast substantially decreased their ability to grow on glycine as the sole nitrogen source. Cells were grown in SD medium containing 30 g/l glycine as nitrogen source. Growth was determined by optical density (OD) of the culture at 600 nm. Data shown are the mean ± SEM for four replicates for wild type and <i>lpd1</i>Δ cells and nine replicates for <i>hem25</i>Δ cells. C) Serine is synthesized from the glycolytic intermediate 3-phosphoglycerate through a series of reactions that includes phosphoserine transaminase (PSAT1 in humans, Ser1 in <i>S</i>. <i>cerevisiae</i>). Serine is normally the main source of one carbon units (CH<sub>2</sub>-THF, 5,10 methylenetetrahydrofolate and its metabolites) in cells. Inactivation of the <i>SER1</i> gene in yeast results in yeast cells that are auxotrophic for serine. Glycine supplementation can also overcome a mutation in the <i>SER1</i> gene as glycine can serve as a metabolic source for both serine and one carbon units. However, this capacity depends entirely on mitochondrial glycine import. The import of glycine into the mitochondria can generate one carbon units in the form of CH<sub>2</sub>-THF through the activity of the glycine cleavage system (GCV). In addition, mitochondrial serine hydroxymethyltransferase (SHMT2 in humans, Shm1 in yeast) catalyzes the synthesis of serine from glycine and CH<sub>2</sub>-THF, with serine exported into the cytoplasm to be consumed for several anabolic pathways including the synthesis of CH<sub>2</sub>-THF. Simultaneously, CH<sub>2</sub>-THF generated from glycine is oxidized to formate and also exported into the cytoplasm as a source of cytoplasmic one carbon units. D) An inability to import glycine into the mitochondria prevents glycine supplementation from providing serine and one carbon units to cells with an inactivated <i>SER1</i> gene. This was found to be the case upon inactivation of the yeast <i>HEM25</i> gene. Cells were grown to mid-log phase in SD medium containing 1 mM serine, and 1:10 serial dilutions plated on SD medium with no supplements or supplemented with serine or glycine.</p
Rescue of growth and heme content in yeast models of congentical sideroblastic anemia.
<p>A) Yeast cells were grown to mid-log phase in the absence or presence of 5 mM glycine, 1 mM serine or 50 μg/ml 5-Ala (the <i>hem1</i>Δ strain is normally grown in the presence of 0.5 μg/ml 5-Ala to allow for growth at a wild type rate) and cells were processed for heme determination. Heme values are the mean ± SEM of at least six independent determinations. B) Yeast cells of the indicated genotypes were grown to mid-log phase and processed for heme determination. C) Cells were grown to mid-log phase in SD medium containing 1 mM serine, and 1:10 serial dilutions plated on SD medium supplemented with glycine, 5-Ala, or serine. D) Cells were grown to mid-log phase in the absence or presence of 5 mM glycine, 1 mM serine or 50 μg/ml 5-Ala and processed for heme determination.</p