Molecular and biochemical features of the mitochondrial enzyme ornithine transcarbamylase: a possible new role as a signaling factor.

Ornithine transcarbamylase (OTC; EC 2.1.3.3) is a one-carbon-unit transferring enzyme that synthesizes citrulline using ornithine and carbamoylphosphate as substrates. It is involved in the metabolic transformation of arginine and proline, and it participates in the urea cycle in vertebrates and in the formation of putrescine in plants. Its enzymatic reaction is consistent with a ping-pong mechanism. OTC is expressed in a large variety of organisms from bacteria to mammals. Its gene can be regulated by glucocorticoids and other transcriptional factors such as C/EBP and HNF-4. The functional enzyme exists mostly as a trimer with an approximate molecular weight of 38 kDa. Inborn errors associated with a deficiency of OTC activity cause mainly urea cycle-related disorders, and lead to hyperammonemic states that may become lethal. In humans and experimental animals, OTC is localized in the mitochondrial matrix, mainly in the liver, but it is also in the intestinal epithelial cells. Some states of hepatotoxicity are associated with hepatocyte disruption and release of OTC into the bloodstream. However, recent evidence suggests that during active cell proliferation (e.g., during liver regeneration), OTC is also released from the hepatic tissue but without apparent damage. In this situation, extracellular and circulating hepatic OTC could be playing a different role, possibly functioning as a signaling molecule.

AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice.

Ornithine transcarbamylase (OTC) deficiency, the most common urea cycle disorder, is associated with severe hyperammonemia accompanied by a high risk of neurological damage and death in patients presenting with the neonatal-onset form. Contemporary therapies, including liver transplantation, remain inadequate with considerable morbidity, justifying vigorous investigation of alternate therapies. Clinical evidence suggests that as little as 3% normal enzyme activity is sufficient to ameliorate the severe neonatal phenotype, making OTC deficiency an ideal model for the development of liver-targeted gene therapy. In this study, we investigated metabolic correction in neonatal and adult male OTC-deficient Spf(ash) mice following adeno-associated virus (AAV)2/8-mediated delivery of the murine OTC complementary DNA under the transcriptional control of a liver-specific promoter. Substantially supraphysiological levels of OTC enzymatic activity were readily achieved in both adult and neonatal mice following a single intraperitoneal (i.p.) injection, with metabolic correction in adults being robust and life-long. In the neonates, however, full metabolic correction was transient, although modest levels of OTC expression persisted into adulthood. Although not directly testable in Spf(ash) mice, these levels were theoretically sufficient to prevent hyperammonemia in a null phenotype. This loss of expression in the neonatal liver is the consequence of hepatocellular proliferation and presents an added challenge to human therapy.

Glycosylation variant analysis of recombinant human tissue plasminogen activator produced in urea-cycle-enzyme-expressing Chinese hamster ovary (CHO) cell line.

Tissue plasminogen activator (tPA) was produced in ornithine transcarbamoylase (OTC) cells by introducing the tPA gene into OTC cells. OTC cells were originally derived from Chinese hamster ovary (CHO) cells and express the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and OTC. To investigate glycosylation variants, tPA variants produced in serum-supplemented culture medium of OTC-tPA cells were separated by lysine-Sepharose 4B chromatography. Unlike in previous studies that used lysine-Sepharose chromatography, two peaks were identified to correspond to eluted glycosylation variants type I and II and type II and the percentages of the type I and type II variants were found to be 23% and 77%, respectively. The biological activities of the type I and II and type II variants were twofold that of the Third International tPA Standard (98/714) produced in the CHO cell line, and the activity of type II variant was 12.6% higher than that of the type I and II variants. These results demonstrate that tPA produced in urea-cycle-enzyme-producing OTC cells have a very high biological activity and the percentage of type II variant which is very valuable for the biopharmaceutical industry is higher than that of any report using CHO cells.

Differentiation effects by the combination of spheroid formation and sodium butyrate treatment in human hepatoblastoma cell line (Hep G2): a possible cell source for hybrid artificial liver.

The aim of this study was to investigate the feasibility of human hepatoblastoma cell line (Hep G2), which differentiates by spheroid formation, and treatment with sodium butyrate (SB) as a cell source for hybrid artificial liver (HAL). Hep G2 spontaneously formed spheroids in polyurethane foam (PUF) within 3 days of culture and restored weak ammonia removal activity. Treatment with SB, which is a histone deacetylase inhibitor, further increased the ammonia removal activity of Hep G2 spheroids in a concentration-dependent manner. The activation of ornithine transcarbamylase--a urea cycle enzyme--was significantly related to the upregulation of ammonia removal by spheroid formation, but scarcely contributed to the further upregulation following SB treatment. In contrast with ammonia removal, treatment with SB reduced the albumin secretion of Hep G2 spheroids in a concentration-dependent manner. In the PUF-HAL module in a circulation culture, the ammonia removal rate and albumin secretion rate (per unit volume of the module) of Hep G2 spheroids treated with 5 mM SB were almost the same as those of primary porcine hepatocyte spheroids. These results suggest that simultaneous use of spheroid formation and SB treatment in Hep G2 is beneficial in enhancing the functions of human hepatocytes with potential applications in regenerative medicine and drug screening.

Disruption of the Aspergillus niger argB gene: a tool for transformation.

We disrupted the Aspergillus niger gene argB, encoding ornithine transcarbamylase. Full characterisation of the argB deletion was performed by Southern blot analysis, growth tests and by means of mitotic recombination, complementation and transformation. The argB locus was found to be physically removed, thus creating an auxotrophic mutation. The latter can be supplemented by addition of arginine into the culture medium. The argB gene and its disruption do not correlate to the argI13 (formerly argB13) allele described. The delta argB is on chromosome I whereas argI13 is on V. In addition, the argI13 mutation can only be complemented by the A. nidulans argB gene, whereas the new argB deletion can be complemented by both the A. niger and A. nidulans argB genes. The delta argB strain has been used to generate several strains in a breeding programme and to study the expression of important genes, such as areA and kexB.

Determining the relative rates of change for prokaryotic and eukaryotic proteins with anciently duplicated paralogs.

The relative rates of change for eight sets of ubiquitous proteins were determined by a test in which anciently duplicated paralogs are used to root the universal tree and distances are calculated between each taxonomic group and the last common ancestor. The sets included ATPase subunits, elongation factors, signal recognition particle and its receptor, three sets of tRNA synthetases, transcarbamoylases, and an internal duplication in carbamoyl phosphate synthase. In each case phylogenetic trees were constructed and the distances determined for all pairs. Taken over the period of time since their last common ancestor, average evolutionary rates are remarkably similar for Bacteria and Eukarya, but Archaea exhibit a significantly slower average rate.

Restoration of nitrogen homeostasis in a man with ornithine transcarbamylase deficiency.

We evaluated the hypothesis that sodium phenylbutyrate-induced phenylacetylglutamine biosynthesis in a man with partial ornithine transcarbamylase (OTC) deficiency has a dual effect; it provides an additional vehicle for waste nitrogen excretion, and in the process it suppresses the patient's residual urea N synthesis, which then may be available for N homeostasis if the need arises. A 38-year-old man was studied over three periods. Period I was a control period during which he received a fixed caloric and N intake plus L-citrulline. Phenylbutyrate was added in period II and was maintained during period III, during which his N intake was increased. Plasma levels of ammonium and glutamine and net urea N synthesis were measured in each period; phenylacetylglutamine N synthesis was measured in periods II and III. These studies demonstrated that phenylbutyrate administration led to a 73% decrease in net de novo urea N synthesis during period II, which subsequently increased threefold in period III in response to the increased N intake. Phenylacetylglutamine N synthesis was 2.27 g/d, similar to his estimated maximum net urea N synthesis of 2.65 g/d. During periods II and III, his plasma levels of ammonium and glutamine improved as compared with period I when they were abnormally high. We conclude that sodium phenylbutyrate treatment of patients with urea cycle disorders who have significant residual enzyme activity results in both an improvement in waste N excretion and improved N homeostasis as a result of the generation of a reserve urea N synthetic capacity. This therapeutic approach may be useful in other nitrogen accumulation decreases, eg, portal-systemic encephalopathy.

Effect of L-carnitine on cerebral and hepatic energy metabolites in congenitally hyperammonemic sparse-fur mice and its role during benzoate therapy.

Sparse-fur (spf) mutant mice with X-linked ornithine transcarbamylase deficiency were used to study the effect of L-carnitine on energy metabolites in congenital hyperammonemia. L-Carnitine was used at doses of 2, 4, 8, or 16 mmol/kg body weight (BW), and levels of ammonia, glutamine, glutamate, and some intermediates of energy metabolism were measured in brain and liver of spf/Y mice. Cerebral and hepatic levels of ammonia were decreased with 4 mmol L-carnitine (P < .001), whereas other doses did not seem to have any effect on this metabolite. Cerebral levels of glutamine were decreased following administration of L-carnitine at doses of up to 4 mmol/kg BW, whereas hepatic glutamine levels remained unaltered at all doses of L-carnitine. Both cerebral and hepatic levels of pyruvate, lactate, and alpha-ketoglutarate were decreased at doses of up to 8 mmol L-carnitine/kg BW. L-Carnitine treatment elevated adenosine triphosphate (ATP), free coenzyme A (CoA), and acetyl CoA levels in both brain and liver of spf/Y mice. Cytosolic and mitochondrial redox ratios of spf/Y mice, which were altered by congenital chronic hyperammonemia, were partially corrected by L-carnitine administration. L-Carnitine supplementation to spf/Y mice during sodium benzoate therapy also restored the availability of free CoA and ATP, thus counteracting the adverse effects of higher doses of sodium benzoate. These changes in free CoA and acetyl CoA levels could be due to the deinhibition of pantothenate kinase and stimulation of fatty acid oxidation by L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)