5-aminolevulinate Synthase Research Articles

Metabolic engineering of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid.

5-Aminolevulinic acid (5-ALA) has recently attracted attention for its potential applications in the fields of medicine and agriculture. In this study, Corynebacterium glutamicum was firstly engineered for 5-ALA production via the C4 pathway. HemA encoding 5-aminolevulinic acid synthase from Rhodobacter sphaeroides was codon optimized and expressed in C. glutamicum ATCC13032, resulting in accumulation of 5-ALA. Deletion of all known genes responsible for the formation of acetate and lactate further enhanced production of 5-ALA. Overexpression of ppc gene encoding phoenolpyruvate carboxylase resulted in an accumulation of 5-ALA up to 2.06 ± 0.05 g/L. Furthermore, deletion of high-molecular-weight penicillin-binding proteins (HMW-PBPs) genes pbp1a, pbp1b, and pbp2b led to an increase in 5-ALA production of 13.53%, 29.47%, and 22.22%, respectively. Finally, 5-ALA production was enhanced to 3.14 ± 0.02 g/L in shake flask by heterologously expressing rhtA encoding threonine/homoserine exporter, and 86.77% of supplemented glycine was channeled toward 5-ALA production in shake flask. The engineered C. glutamicum ALA7 strain produced 7.53 g/L 5-ALA in a 5 L bioreactor. This study demonstrated the potential utility of C. glutamicum as a platform for metabolic production of 5-ALA. Change of cell permeability by metabolic engineering HMW-PBPs may provide a new strategy for biochemicals production in Corynebacterium glutamicum. Biotechnol. Bioeng. 2016;113: 1284-1293. © 2015 Wiley Periodicals, Inc.

Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction

5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO2, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn-85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function, and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. Although wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn-150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, thus ensuring that ALA is predominantly available for heme biosynthesis.

Activity of Heme Synthesis Enzymes in the Bone Marrow and Liver of August and Wistar Rats During the Neonatal Period and After Acute Postnatal Hypoxia.

Activity of heme synthesis enzymes in newborn August and Wistar rats was studied after acute hypoxic hypoxia. Daily production of erythrocytes and activities of aminolevulinate synthase, aminolevulinate dehydratase, and heme synthetase were measured in the bone marrow (15-30 min after birth and on days 1 and 3 of life) and liver (day 3 after birth). Hypoxia was followed by a decrease in activity of heme synthesis enzymes in the liver (especially in August rats) and reduction of the daily erythrocyte production (especially in Wistar rats). Our results suggest that the response of heme synthesis enzymes to hypoxic exposure in newborn rats is genetically determined. The observed changes are more pronounced in Wistar rats.

MiR-218 Inhibits Erythroid Differentiation and Alters Iron Metabolism by Targeting ALAS2 in K562 Cells.

microRNAs (miRNAs) are involved in a variety of biological processes. The regulatory function and potential role of miRNAs targeting the mRNA of the 5′-aminolevulinate synthase 2 (ALAS2) in erythropoiesis were investigated in order to identify miRNAs which play a role in erythroid iron metabolism and differentiation. Firstly, the role of ALAS2 in erythroid differentiation and iron metabolism in human erythroid leukemia cells (K562) was confirmed by ALAS2 knockdown. Through a series of screening strategies and experimental validations, it was identified that hsa-miR-218 (miR-218) targets and represses the expression of ALAS2 by binding to the 3′-untranslated region (UTR). Overexpression of miR-218 repressed erythroid differentiation and altered iron metabolism in K562 cells similar to that seen in the ALAS2 knockdown in K562 cells. In addition to iron metabolism and erythroid differentiation, miR-218 was found to be responsible for a reduction in K562 cell growth. Taken together, our results show that miR-218 inhibits erythroid differentiation and alters iron metabolism by targeting ALAS2 in K562 cells.

WITHDRAWN: Over-expression 5-aminolevulinic acid synthase 2 in nonerythroid cell may causes protoporphyrin IX accumulation

Protoporphyrin IX (PpIX) has been used as an efficient sensitizer in photodynamic diagnose, photodynamic therapy, and sonodynamic therapy. The level of PpIX is very important for diagnose or therapy effects. 5-aminolevulinic acid synthase 2 (ALAS2) is the key enzyme upstream of PpIX synthesis. To increase PpIXaccumulation, ALAS2 overexppression transgenic mice were generated. Plasmid containing alas2 gene was transfected in colonic carcinoma cell lines. Both in tissues of transgenic mice and in colonic carcinoma cells, the amount of PpIXaccumulation did increased. At the meanwhile, level of heme, which down stream of PpIX had not been changed. Overexpression ALAS2 in nonerythriod cells may become a novel approach to cause PpIX accumulation.

Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release.

Regulation of 5-aminolevulinate synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically, and thermodynamically. Enhanced activities of the XLPP variants resulted from increases in the rate at which the product 5-aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5'-phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon binding of ALA to the XLPP variants demonstrated that the protein conformational transition step associated with product release was predominantly affected. Of relevance is the fact that XLPP could also be modeled in cell culture. We propose that (1) the XLPP mutations destabilize the succinyl-CoA-induced hALAS2 closed conformation and thus accelerate ALA release, (2) the extended C-terminus of wild-type mammalian ALAS2 provides a regulatory role that allows for allosteric modulation of activity, thereby controlling the rate of erythroid heme biosynthesis, and (3) this control is disrupted in XLPP, resulting in porphyrin accumulation.

Purification and functional characterization of thermostable 5-aminolevulinic acid synthases.

As 5-aminolevulinic acid synthase (ALAS), the key enzyme for 5-aminolevulinic acid (ALA) synthesis, is unstable, we have sought to find thermostable ALASs from thermophilic organisms. Three ALASs from thermophiles Geobacillus thermoglucosidasius (GT-ALAS), Laceyella sacchari (LS-ALAS) and Pseudomonas alcaliphila (PA-ALAS) were purified and characterized. All enzymes were more stable than two previously studied ALASs from Rhodopseudomonas palustris and Rhodobacter sphaeroides. There was almost no activity change after 60h at 37°C for the three thermostable enzymes. This contrasts with the other two enzymes which lost over 90% activities in just 1h. Furthermore, the specific activity of LS-ALAS (7.8Umg(-1)) was also higher than any previously studied ALASs. Thermostable ALASs were found in thermophilic organisms and this paves the way for developing cell free processes for enzymatic production of ALA from bulk chemicals succinate and glycine.

Identification of the Mitochondrial Heme Metabolism Complex.

Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex.

Inducing iron deficiency improves erythropoiesis and photosensitivity in congenital erythropoietic porphyria

AbstractCongenital erythropoietic porphyria (CEP) is an autosomal recessive disorder of heme synthesis characterized by reduced activity of uroporphyrinogen III synthase and the accumulation of nonphysiologic isomer I porphyrin metabolites, resulting in ineffective erythropoiesis and devastating skin photosensitivity. Management of the disease primarily consists of supportive measures. Increased activity of 5-aminolevulinate synthase 2 (ALAS2) has been shown to adversely modify the disease phenotype. Herein, we present a patient with CEP who demonstrated a remarkable improvement in disease manifestations in the setting of iron deficiency. Hypothesizing that iron restriction improved her symptoms by decreasing ALAS2 activity and subsequent porphyrin production, we treated the patient with off-label use of deferasirox to maintain iron deficiency, with successful results. We confirmed the physiology of her response with marrow culture studies.

A method for determining δ-aminolevulinic acid synthase activity in homogenized cells and tissues

ObjectiveIn mammalian cells the rate-limiting step in heme biosynthesis is the formation of δ-aminolevulinic acid (ALA). The reaction intermediates, porphyrins and iron and the final product, heme can be highly cytotoxic if allowed to accumulate. The importance of maintaining the levels of metabolic intermediates and heme within a narrow range is apparent based on the complex homeostatic system(s) that have developed. Ultimately, determining the enzymatic activity of ALA synthase (ALAS) present in the mitochondria is highly beneficial to confirm the effects of the transcriptional, translational and post-translational events. The aim of this study was to develop a highly sensitive assay for ALAS that could be used on whole tissue or cellular homogenates. Design and methodsA systematic approach was used to optimize steps in formation of ALA by ALAS. Reducing the signal to noise ratio for the assay was achieved by derivatizing the ALA formed into a fluorescent product that could be efficiently separated by ultra performance liquid chromatography (UPLC) from other derivatized primary amines. The stability of ALAS activity in whole tissue homogenate and cellular homogenate was determined after extended storage at −80°C. ConclusionsA method for assaying ALAS has been developed that can be used with tissue homogenates or cellular lysates. There is no need to purify mitochondria and radiolabeled substrates are not needed for this assay. General laboratory reagents can be used to prepare the samples. Standard UPLC chromatography will resolve the derivatized ALA peak. Samples of tissue homogenate can be stored for approximately one year without significant loss of enzymatic activity.

The Mtm1p carrier and pyridoxal 5′-phosphate cofactor trafficking in yeast mitochondria

Biochemical communication between the cytoplasmic and mitochondrial subsystems of the cell depends on solute carriers in the mitochondrial inner membrane that transport metabolites between the two compartments. We have expressed and purified a yeast mitochondrial carrier protein (Mtm1p, YGR257cp), originally identified as a manganese ion carrier, for biochemical characterization aimed at resolving its function. High affinity, stoichiometric pyridoxal 5′-phosphate (PLP) cofactor binding was characterized by fluorescence titration and calorimetry, and the biochemical effects of mtm1 gene deletion on yeast mitochondria were investigated. The PLP status of the mitochondrial proteome (the mitochondrial ‘PLP-ome’) was probed by immunoblot analysis of mitochondria isolated from wild type (MTM1+) and knockout (MTM1−) yeast, revealing depletion of mitochondrial PLP in the latter. A direct activity assay of the enzyme catalyzing the first committed step of heme biosynthesis, the PLP-dependent mitochondrial enzyme 5-aminolevulinate synthase, extends these results, providing a specific example of PLP cofactor limitation. Together, these experiments support a role for Mtm1p in mitochondrial PLP trafficking and highlight the link between PLP cofactor transport and iron metabolism, a remarkable illustration of metabolic integration.

Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification.

The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.

Transcriptional regulation of genes related to progesterone production.

Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert Δ(5)-Δ(4) isomerization activity to produce progesterone in a cooperative fashion with 3β-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production.

Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis

5-Aminolevulinate synthase (ALAS) catalyzes the initial step of mammalian heme biosynthesis, the condensation between glycine and succinyl-CoA to produce CoA, CO2, and 5-aminolevulinate. The crystal structure of Rhodobacter capsulatus ALAS indicates that the adenosyl moiety of succinyl-CoA is positioned in a mainly hydrophobic pocket, where the ribose group forms a putative hydrogen bond with Lys156. Loss-of-function mutations in the analogous lysine of human erythroid ALAS (ALAS2) cause X-linked sideroblastic anemia. To characterize the contribution of this residue toward catalysis, the equivalent lysine in murine ALAS2 was substituted with valine, eliminating the possibility of a hydrogen bond. The K221V substitution produced a 23-fold increase in the KmSCoA and a 97% decrease in kcat/KmSCoA. This reduction in the specificity constant does not stem from lower affinity toward succinyl-CoA, since the KdSCoA of K221V is lower than that of wild-type ALAS. For both enzymes, the KdSCoA value is significantly different from the KmSCoA. That K221V has stronger binding affinity for succinyl-CoA was further deduced from substrate protection studies, as K221V achieved maximal protection at lower succinyl-CoA concentration than wild-type ALAS. Moreover, it is the CoA, rather than the succinyl moiety, that facilitates binding of succinyl-CoA to wild-type ALAS, as evident from identical KdSCoA and KdCoA values. Transient kinetic analyses of the K221V-catalyzed reaction revealed that the mutation reduced the rates of quinonoid intermediate II formation and decay. Altogether, the results imply that the adenosyl-binding site Lys221 contributes to binding and orientation of succinyl-CoA for effective catalysis.

Acute porphyria: The drug demon etiology

Drugs are one of the important factors that precipitate acute attacks in patients with porphyria. Various in vitro and preclinical animal models exist for determining porphyrogenicity of a drug, but these predictions are not very reliable. Ambiguities still remain with many drugs as to whether they are safe in patients with porphyria. An algorithm for determining the porphyrogenic potential using evidence from anectodotal reports, lipophilicity, affinity of a drug to the Cytochrome enzymes (CYP450), ability to induce or irreversibly inhibit either CYP450 2C9 or CYP450 3A4 and the ability to induce hepatic 5-aminolevulinate synthase has been devised. Management of an acute attack of porphyria involves symptomatic treatment and administration of haem arginate. Several international networks have been initiated that serve as good sources of information for the safe use of drugs to be used in porphyria.

Analysis of Heme Biosynthetic Pathways in a Recombinant Escherichia coli.

Bacterial heme was produced from a genetic-engineered Escherichia coli via the porphyrin pathway and it was useful as an iron resource for animal feed. The amount of the E. colisynthesized heme, however, was only few milligrams in a culture broth and it was not enough for industrial applications. To analyze heme biosynthetic pathways, an engineered E. coli artificially overexpressing ALA synthase (hemA from Rhodobacter sphaeroides) and pantothenate kinase (coaA gene from self geneome) was constructed as a bacterial heme-producing strain, and both the transcription levels of pathway genes and the intermediates concentrations were determined from batch and continuous cultures. Transcription levels of the pathway genes were not significantly changed among the tested conditions. Intracellular intermediate concentrations indicated that aminolevulinic acid (ALA) and coenzyme A (CoA) were enhanced by the hemA-coaA co-expression. Intracellular coproporphyrinogen I and protoporphyrin IX accumulation suggested that the bottleneck steps in the heme biosynthetic pathway could be the spontaneous conversion of HMB to coproporphyrinogen I and the limited conversion of protoporphyrin IX to heme, respectively. A strategy to increase the conversion of ALA to heme is discussed based on the results.

Impact of TET2 Deficiency on Iron Metabolism in Erythroblasts: A Potential Link to Ring Sideroblast Formation

Background: Sideroblastic anemia is characterized by the presence of ring sideroblasts (RS) in the bone marrow (BM). RS are caused by iron accumulation in the mitochondria of erythroblasts and are seen in both acquired and congenital forms of sideroblastic anemia. Although dysfunction in mitochondrial metabolism has been implicated in the pathogenesis of sideroblastic anemia, the true mechanism leading to RS formation remains elusive. Clonal sideroblastic anemia is usually acquired in the context of myelodysplastic syndrome (MDS): The presence of 15% or more RS in the BM with appropriate morphologic and cytogenetic criteria for MDS is best classified as refractory anemia with ring sideroblasts (RARS), but varying quantities of RS (<15%) can also occur in refractory anemia with multilineage dysplasia (RCMD) and other myeloid malignancies as well. Intriguingly, a subset of MDS as well as myeloid malignancies has been reported to harbor a somatic mutation of the TET2gene (Delhommeau F et al., 2009; Malcovati et al., 2014), which regulates DNA demethylation by hydroxylating 5-methylcytosine to 5-hydroxymethylcytosine. Thus, because TET2 has also been shown to play an important role during erythroid differentiation (Pronier et al., 2011), TET2 may be involved in iron metabolism and/or heme biosynthesis in erythroblasts, contributing to the formation of RS. To explore this possibility, we conducted biological and molecular analyses based on TET2-knockdown mice.Methods: TET2-knockdown mice (Ayu17-449, Tet2trap) were generated by gene trap project (Shide et al., 2012). Four-month-old heterozygous Tet2trap/+ mice were used in the present study. Serum levels of iron, total iron-binding capacity, unbound-iron binding capacity and ferritin were measured by enzyme-linked immunosorbent assay. BM cells were stained for CD71 and Ter119 (BD Pharmingen, San Jose, CA, USA), and sorted into I–IV populations according to a previously reported method (Socolovsky et al., 2001). To obtain Ter119+ erythroblasts, a magnetic cell sorting system was used (Miltenyi Biotec, Auburn, CA, USA). Western blot analysis was performed with anti-mitochondrial ferritin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Prussian blue staining was used to assess iron deposition in erythroblasts. DNA methylation status was assessed with the EpiXploreTM Methylated DNA Enrichment Kit (Clontech, Mountain View, CA, USA). Statistical significance was assessed by the two-sided ttest, and p value of <0.05 were considered statistically significant.Results: Tet2trap/+ mice at 4 months exhibited mild normocytic anemia in comparison to the wild-type (WT) mice (hemoglobin; 14.5 ± 1.1 and 13.2 ± 0.6 g/dl, for WT and Tet2trap/+, respectively, p < 0.05, n = 8). Concomitantly, the number of cells in erythroid populations III–IV (polychromatic to orthochromatic erythroblasts) was decreased. Interestingly, serum iron and ferritin levels were significantly elevated in Tet2trap/+ mice (ferritin; 104.7 ± 42.4 and 171.6 ± 89.5 ng/ml, for WT and Tet2trap/+, respectively, p < 0.05, n = 8). Western blot analysis confirmed that the amount of mitochondrial ferritin was increased in Ter119+ erythroblasts from Tet2trap/+ mice compared with those from WT mice. On the other hand, BM from Tet2trap/+ mice did not show an increased number of RS. To explore the molecular mechanism by which TET2 deficiency induces iron overload, quantitative reverse transcription–polymerase chain reaction analysis was conducted with Ter119+ cells for genes involved in erythroid differentiaition, heme biosynthesis and iron metabolism. The analysis demonstrated significant downregulation of heme oxygenase 1 (Hmox1), ferrochelatase (Fech) and Tet2 in Tet2trap/+ mice, whereas the expression of erythroid-specific 5-aminolevulinate synthase (Alas2), mitoferrin (Slc25a37) and Gata1 were unchanged. Because a CpG island was idenitified in the promoter of Fech (http://genome.ucsc.edu), we evaluated its DNA methylation status and found that the CpG site of Fech shows significantly high methylation in Ter119+ cells of Tet2trap/+ mice compared with those of WT mice. As FECH catalyzes the insertion of ferrous iron into the protoporphyrin IX to produce heme, the reduced expression of Fech by TET2 deficiency may result in inhibiton of heme synthesis, leading to iron overload in mitochondria.Conclusion: TET2 is involved in iron and heme metabolism in erythroblasts. DisclosuresFujiwara:Chugai Pharmaceutical CO., LTD.: Research Funding.

Exploring the Potential Usefulness of 5-Aminolevulinic Acid for X-Linked Sideroblastic Anemia

(Background) Congenital sideroblastic anemia (CSA) is an inherited disease; it is a microcytic type of anemia characterized by bone marrow sideroblasts with excess iron deposition in the mitochondria. The most common form of CSA is XLSA (X-linked sideroblastic anemia), which is attributed to mutations in the X-linked gene ALAS2 (erythroid-specific 5-aminolevulinate synthase). ALAS2 encodes the first and rate-limiting enzyme involved in heme biosynthesis in erythroid cells, which utilizes glycine and acetyl-coenzyme A to form 5-aminolevulinic acid (ALA) and also requires pyridoxal 5′-phosphate (PLP, vitamin B6) as a cofactor. Based on the evidence that half of the XLSA cases were unresponsive to PLP (Ohba et al. Ann Hematol 2013), ALA supplementation could emerge as an alternative therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. As a preclinical study, we focused our study on the effect of ALA on human erythroid cells. Furthermore, we investigated the molecular mechanism by which ALA is transported into erythroid cells.(Method) Human K562 erythroid cells as well as human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells (Kurita et al. PLoS ONE 2013) were used for the analysis. We investigated the effects of ALA (0.01, 0.1, and 0.5 mM for 72 h) on heme content, hemoglobinization, and erythroid-related gene expression. Heme content was determined fluorometrically at 400 nm (excitation) and 662 nm (emission). Small interfering RNA (siRNA)-mediated knockdown of ALAS2 was conducted using Amaxa Nucleofector™ (Amaxa Biosystems, Koln, Germany). For transcription profiling, Human Oligo chip 25K (Toray, Tokyo, Japan) was used for control and ALAS2 siRNA-treated HiDEP cells. Gamma-aminobutyric acid (GABA) (Sigma, St. Louis, MO, USA) was used at concentrations of 10 and 20 mM.(Results) First, we demonstrated that ALA treatment resulted in significant dose-dependent accumulation of heme in K562 cells. Concomitantly, the treatment substantially induces erythroid differentiation as assessed using hemoglobin (benzidine) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes such as the globin genes (HBA and HBG) and the heme oxygenase 1 (HMOX1) gene in K562 cells. To investigate the mechanism by which ALA was transported into erythroid cells, we conducted quantitative RT-PCR analysis for previously reported ALA transporters, including SLC15A1, SLC15A2, SLC36A1, and SLC6A13 (Frølund et al. Br J Pharmacol 2010; Ahlin et al. Drug Metab Dispos 2009; Moretti et al. Br J Cancer 2002). The analysis revealed that SLC36A1 was abundantly expressed in K562 and HiDEP cells. Thus, GABA was added to K562 cells to competitively inhibit SLC36A1-mediated transport (Frølund et al. Br J Pharmacol 2010). GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells.Next, siRNA-mediated knockdown of ALAS2 in HiDEP cells resulted in a significant decrease in the expression of globin genes as well as HMOX1; however, ringed sideroblasts were not observed. Microarray analysis revealed >2-fold up- and down-regulation of 38 and 68 genes caused by ALAS2 knockdown, respectively. The downregulated gene ensemble included globins (HBZ, HBG, HBE, HBD, and HBM) as well as genes involved in iron metabolism (ferritin heavy chain 1: FTH1, transferrin receptor: TFRC and glutaredoxin-1: GLRX5). Gene ontology analysis revealed significant enrichment of cellular iron ion homeostasis (p = 0.000076), cell division (p = 0.00062), DNA repair (p = 0.0006) and translation (p = 0.018), implying that heme was involved in various biological processes in erythroid cells. Interestingly, ALA treatment significantly improved the consequences of ALAS2 knockdown-mediated downregulation of HBA, HBG, and HMOX1.(Conclusion) ALA appears to enter into erythroid cells mainly by SLC36A1 and utilized to generate heme precursor. Thus,ALA may represent a novel therapeutic option for CSA, particularly for cases harboring ALAS2 mutations. DisclosuresFujiwara:Chugai Pharmaceutical, CO., LTD.: Research Funding.

Aminolevulinate synthase 2 mediates erythrocyte differentiation by regulating larval globin expression during Xenopus primary hematopoiesis

Hemoglobin synthesis by erythrocytes continues throughout a vertebrate’s lifetime. The mechanism of mammalian heme synthesis has been studied for many years; aminolevulinate synthase 2 (ALAS2), a heme synthetase, is associated with X-linked dominant protoporphyria in humans. Amphibian and mammalian blood cells differ, but little is known about amphibian embryonic hemoglobin synthesis. We investigated the function of the Xenopus alas2 gene (Xalas2) in primitive amphibian erythrocytes and found that it is first expressed in primitive erythroid cells before hemoglobin alpha 3 subunit (hba3) during primary hematopoiesis and in the posterior ventral blood islands at the tailbud stage. Xalas2 is not expressed during secondary hematopoiesis in the dorsal lateral plate. Hemoglobin was barely detectable by o-dianisidine staining and hba3 transcript levels decreased in Xalas2-knockdown embryos. These results suggest that Xalas2 might be able to synthesize hemoglobin during hematopoiesis and mediate erythrocyte differentiation by regulating hba3 expression in Xenopus laevis.

The Heme Biosynthesis Pathway Is Essential for Plasmodium falciparum Development in Mosquito Stage but Not in Blood Stages

Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and is proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knock-out parasite lines, lacking 5-aminolevulinic acid synthase and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 5-[(13)C4]aminolevulinic acid, and this assay confirmed that de novo heme synthesis was ablated in FC knock-out parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs.