A single amino acid substitution within the mature sequence of ornithine aminotransferase obstructs mitochondrial entry of the precursor.

We describe here evidence of congenital enzyme mistargeting induced not by abnormalities in the signal sequence. We examined the molecular mechanism of hereditary ornithine aminotransferase (OAT) deficiency causing gyrate atrophy of the choroid and retina (GACR). Nucleotide sequencing of OAT cDNA generated from a GACR patient's mRNA revealed a single base change from C to G at position 268, resulting in an amino acid substitution of neutral Gln(CAA) with negatively charged Glu(GAA) at position 90 (Q90E). Immunohistochemical and transient expression analyses suggested expression of a defective labile OAT in the patient's tissues. However, high-level expression and immunocytochemical analyses elucidated that Q90E OAT (the patient's OAT) was localized within the limits of cytoplasmic free ribosomes in precursor form without any mitochondrial entry, indicating that the patient's precursor OAT was synthesized and rapidly degraded because of accumulation in the cytosol. It is interesting that, although the mutation site (Q90E) in this GACR patient's OAT was within the coding sequence of the mature protein, the precursor exhibited loss of mitochondrial targeting function. These findings suggest that not only the signal sequence but a critical part of the mature sequence plays an essential role in mitochondrial entry of the OAT precursor protein.     

Ornithine delta-aminotransferase mutations in gyrate atrophy. Allelic heterogeneity and functional consequences.

Ornithine delta-aminotransferase is a nuclear-encoded mitochondrial matrix enzyme which catalyzes the reversible interconversion of ornithine and alpha-ketoglutarate to glutamate semialdehyde and glutamate. Inherited deficiency of ornithine delta-aminotransferase results in ornithine accumulation and a characteristic chorioretinal degeneration, gyrate atrophy of the choroid and retina. We have surveyed the ornithine delta-aminotransferase genes of gyrate atrophy patients for mutations. Using a variety of techniques, we discovered and molecularly characterized 21 newly recognized ornithine delta-aminotransferase alleles. We determined the consequences of these and three previously described mutations on ornithine delta-aminotransferase mRNA, antigen, and enzyme activity in cultured fibroblasts. The majority (20/24) of these alleles produce normal amounts of normally sized ornithine delta-aminotransferase mRNA. By contrast, only 2/24 had normal amounts of ornithine delta-aminotransferase antigen. Reproducing these mutations by site-directed mutagenesis and expressing the mutant ornithine delta-aminotransferase in Chinese hamster ovary cells confirms that several of these mutations inactivate ornithine delta-aminotransferase and cause gyrate atrophy in these patients.     

Gyrate atrophy of the choroid and retina: assignment of the ornithine aminotransferase structural gene to human chromosome 10 and mouse chromosome 7.

Gyrate atrophy of the choroid and retina is an autosomal recessive, blinding human disease caused by a deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT). Since human OAT cDNA hybridizes to DNA sequences on both human chromosomes 10 and X, a locus coding for OAT enzyme activity may be present on one or both of these human chromosomes. We have used a series of mouse-human somatic cell hybrids, in combination with starch gel electrophoresis and a histochemical stain for OAT enzyme activity, to assign the structural gene for OAT to human chromosome 10. Our results suggest that the human X chromosome does not contain a locus coding for OAT enzyme activity. In addition, we have used a panel of Chinese hamster-mouse hybrids to assign the murine Oat structural gene to mouse chromosome 7. Our findings, combined with recent molecular studies, indicate that human OAT probes specific for chromosome 10 will be useful for the diagnosis and genetic counseling of individuals at risk for gyrate atrophy.     

Gyrate atrophy of the choroid and retina: characterization of mutant ornithine aminotransferase and mechanism of response to vitamin B6.

The purpose of this study was to characterize the mutant enzyme in nine patients with gyrate atrophy of the choroid and retina associated with ornithine aminotransferase (OAT) deficiency, to elucidate the mechanism of response to pyridoxine in four pyridoxine-responsive patients, and to determine the extent of genetic heterogeneity in both groups of patients. We have measured the apparent Km for pyridoxal phosphate (PLP) in fibroblast mitochondria and the heat stability of OAT at 45 degrees C in the presence and absence of PLP, using a sensitive radiochemical assay. The apparent Km for PLP was higher in pyridoxine-responsive patients than in nonresponsive patients whose apparent Km for PLP was normal. In contrast, the apparent Km for ornithine was normal in the seven patients studied. Surprisingly, the responsive patient with mildest clinical disease had the highest Km for PLP. However, she had the most stable enzyme, which presumably contributed to her milder phenotype. Western blot analyses of mitochondrial proteins, using antibody to human OAT, indicated clearly detectable OAT protein in pyridoxine-responsive patients and in two of five nonresponders, but low or undetectable levels in the other three patients. These data clarify the mechanism of pyridoxine response and indicate heterogeneity within as well as between the pyridoxine-responsive and the nonresponsive patients with gyrate atrophy.     

Uptake of antitetanus F(ab')2 fragments into eukaryotic cells

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.     

Ornithine ketoacid transaminase deficiency in gyrate atrophy of the choroid and retina.

Gyrate atrophy of the choroid and retina is a chorioretinal degeneration associated with hyperornithinemia with an autosomal recessive mode of inheritance. Cultured skin fibroblasts from five affected patients showed a virtual absence of ornithine ketoacid transaminase (OKT) (L-ornithine:2-oxoacid aminotransferase E.C.2.6.1.13) activity. Fibroblasts from four carrier parents showed a 42%-65% reduction in OKT activity. Increasing the concentration of pyridoxal phosphate (vitamin B6 in the assay media resulted in partial restoration of OKT activity in fibroblasts from one out of five patients studied. We conclude that OKT deficiency is closely associated with the genetic defect in gyrate atrophy of the choroid and retina and that genetic heterogeneity exists in this disease.     

Functional inhibition of cytosolic and mitochondrial aspartate aminotransferase by l-2-amino-4-methoxy-trans-3-butenoic acid in isolated rat hepatocytes and mitochondria

The transaminase inhibitor l-2-amino-4-methoxy-trans-3-butenoic acid (AMB) decreased aspartate aminotransferase activity by approximately two-thirds in isolated rat liver mitohondria incubated with succinate, ammonia, and ornithine. Aspartate production by the mitochondria was unaffected over the 30-min incubation period, indicating that mitochondrial aspartate aminotransferase activity is normally far in excess of that required for maximal rates of aspartate production. In rat hepatocytes incubated with lactate, ammonia, and ornithine the inhibition of both the cytosolic and mitochondrial isozymes of aspartate aminotransferase by AMB was partially blocked by the presence of ammonia and ornithine. When pyruvate was substituted for lactate as a carbon source with isolated hepatocytes, the presence of ammonia and ornithine blocked the inhibition by AMB of the mitochondrial but not the cytosolic isozyme of aspartate aminotransferase. Urea formation by cells incubated with lactate, ammonia, and ornithine was unaffected by AMB unless the cells were preincubated with the inhibitor prior to the addition of substrates. However, urea formation by cells incubated in the presence of pyruvate, ammonia, and ornithine was inhibited strongly by AMB even without preincubation. The results suggest that the stimulation of ureogenesis from ammonia and ornithine by pyruvate involves the cytosolic isozyme of aspartate aminotransferase. In contrast, the stimulation of ureogenesis elicited by lactate primarily involved mitochondrial aspartate aminotransferase.     

Immunocytochemical localization of ornithine transcarbamylase in rat intestinal mucosa. Light and electron microscopic study

We investigated light and electron microscopic localization of ornithine transcarbamylase (OTC) in rat intestinal mucosa. In the immunoblotting assay of OTC-related protein, a single protein band with a molecular weight of about 36,500 is observed in extracts of liver and small intestinal mucosa but is not observed in those of stomach and large intestine. For light microscopy, tissue slices of the digestive system were embedded in Epon and stained by using anti-bovine OTC rabbit IgG and the immunoenzyme technique. For electron microscopy, slices of these and the liver tissues were embedded in Lowicryl K4M and stained by the protein A-gold technique. By light microscopy, the absorptive epithelial cells of duodenum, jejunum, and ileum stained positively for OTC, but stomach, large intestine, rectum, and propria mucosa of small intestine were not stained. Electron microscopy showed that gold particles representing the antigenic sites for OTC were confined to the mitochondrial matrix of hepatocytes and small intestinal epithelial cells. However, the enzyme was detected in mitochondria of neither liver endothelial cells, submucosal cells of small intestine, nor large intestinal epithelial cells. Labeling density of mitochondria in the absorptive epithelial cells of duodenum, jejunum, and ileum was about half of that in liver cells.     

Immunohistochemical study of localization of gamma-glutamyl transpeptidase in the rat brain

Gamma-glutamyl transpeptidase (gamma-GTP) is a membrane-bound enzyme which is known to play a crucial role in active transport of amino acids across membrane barriers. We prepared a monoclonal antibody recognizing specifically rat gamma-GTP and investigated localization of the enzyme in the rat brain by immunohistochemistry with this antibody. The antigen was localized on the ependyma, epithelia of the choroid plexus and microvessels. More precise localization of gamma-GTP was examined with immuno-electron microscopy. The antigen was recognized on the microvilli and cilia of the ependymal cells, microvilli of the choroid epithelial cells and luminal membranes of the vascular endothelial cells.     

Preferential localization of calcium-activated neutral protease in epithelial tissues

Immunohistochemical localization of calcium-activated neutral protease (CANP) in rabbit organs was determined using a monoclonal antibody against CANP. In most organs, epithelial tissues reacted intensely: these tissues include great alveolar and squamous alveolar cells in lung; interlobular artery, vein, and bile duct in liver; small vessels in skeletal muscle; glomeruli, juxtanglomerular cells, distal and collecting tubules in kidney; mucous epithelium in gallbladder; interstitial cells in testis; and cuboidal epithelial cells in brain choroid plexus. On the other hand, hepatocytes, epithelial cells which have ill defined basal lamina, were stained very faintly. These observations suggest that the physiological function of CANP is involved with transport systems in epithelial tissues through basal lamina.     

Factors influencing the activity of ornithine aminotransferase in isolated rat liver mitochondria.

1. The characteristics of ornithine catabolism by the aminotransferase pathway in isolated mitochondria were determined. 2. Ornithine synthesis from glutamate and glutamate gamma-semialdehyde produced by the oxidation of proline was studied. No ornithine was formed in the absence of rotenone. 3. The mechanism of ornithine transport was reinvestigated, and the existence of an ornithine+/H+ exchange system postulated. 4. The kinetics of ornithine transport, ornithine catabolism in intact mitochondria and ornithine aminotransferase activity in solubilized mitochondria were compared. It is concluded that ornithine aminotransferase activity in liver mitochondria is rate-limited by the transport of ornithine across the mitochondrial membrane, and that this enzyme is involved primarily in ornithine degradation rather than ornithine synthesis.     

Nonsense-codon mutations of the ornithine aminotransferase gene with decreased levels of mutant mRNA in gyrate atrophy.

A generalized deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is the inborn error in gyrate atrophy (GA), an autosomal recessive degenerative disease of the retina and choroid of the eye. Mutations in the OAT gene show a high degree of molecular heterogeneity in GA, reflecting the genetic heterogeneity in this disease. Using the combined techniques of PCR, denaturing gradient gel electrophoresis, and direct sequencing, we have identified three nonsense-codon mutations and one nonsense codon-generating mutation of the OAT gene in GA pedigrees. Three of them are single-base substitutions, and one is a 2-bp deletion resulting in a reading frameshift. A nonsense codon created at position 79 (TGA) by a frameshift and nonsense mutations at codons 209 (TAT----TAA) and 299 (TAC----TAG) result in abnormally low levels of OAT mRNA in the patient's skin fibroblasts. A nonsense mutation at codon 426 (CGA----TGA) in the last exon, however, has little effect on the mRNA level. Thus, the mRNA level can be reduced by nonsense-codon mutations, but the position of the mutation may be important, with earlier premature-translation termination having a greater effect than a later mutation.     

Retinal-specific ATP-binding cassette transporter (ABCR/ABCA4) is expressed at the choroid plexus in rat brain

ATP-binding cassette (ABC) transporter A4 is a member of the ABC transporter subfamily A which has been reported to be exclusively expressed in the retina. In contrast, a previous report has suggested a possible relationship between ABCA4 and CNS function. The purpose of the present study was to investigate the localization of ABCA4 mRNA and protein in rat brain. In situ hybridization analysis revealed that ABCA4 mRNA was localized in the lateral ventricles. RT-PCR analysis detected ABCA4 mRNA in isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB). Furthermore, ABCA4 protein was also detected in the isolated rat choroid plexus at about 250 kDa by western blot analysis, and its apparent molecular size was reduced by N-glycosidase F treatment. These results suggest that glycosylated ABCA4 protein is expressed in rat choroid plexus epithelial cells. ABCA4 may play a role in the function of the blood-cerebrospinal fluid barrier and affect CSF conditions.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.     

Caveolin-1 and migration of bone-marrow derived cells in the mouse eye

Bone marrow derived cells (BMDCs) can be found in almost every tissue showing a distinct turnover and density. Since caveolin-1 regulates junction-associated proteins in endothelial and epithelial cells, its role for BMDC was investigated in the eyes of caveolin-1 knock-out mice transplanted with GFP-marked BMDC. Distribution and turnover of BMDC in connective tissues (cornea, iris, ciliary body and choroid) was not altered. The absence of caveolin-1, however, caused a significant decrease of BMDC turnover in cornea epithelium, ciliary epithelium, and in the retina. This finding emphasizes an important, hitherto unknown role of caveolin-1 in neuronal and epithelial tissues.     

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.     

Possible mechanism for the decrease of mitochondrial aspartate aminotransferase activity in ischemic and hypoxic rat retinas.

Glutamate is believed to be an excitatory amino acid neurotransmitter in the retina. Enzymes for glutamate metabolism, such as glutamate dehydrogenase, ornithine aminotransferase, glutaminase, and aspartate aminotransferase (AAT), exist mainly in the mitochondria. The abnormal increase of intracellular calcium ions in ischemic retinal cells may cause an influx of calcium ions into the mitochondria, subsequently affecting various mitochondrial enzyme activities through the activity of mitochondrial calpain. As AAT has the highest level of activity among enzymes involved in glutamate metabolism, we investigated the change of AAT activity in ischemic and hypoxic rat retinas and the protection against such activity by calpain inhibitors. We used normal RCS (rdy+/rdy+) rats. For the in vivo studies, we clamped the optic nerve of anesthetized rats to induce ischemia. In the in vitro studies, the eye cups were incubated with Locke's solution saturated with 95% N2/5% CO2. The activity of cytosolic AAT (cAAT) was about 20% of total activity, whereas mitochondrial AAT (mAAT) was about 75% in rat retina. Ninety minutes of ischemia or hypoxia caused a 20% decrease in mAAT activity, whereas cAAT activity remained unchanged. To examine the contribution of intracellular calcium ions to the degradation of mAAT, we used Ca2+-free Locke's solution containing 1 mM EGTA, ryanodine (Ca2+ channel blocker), and thapsigargin (Ca2+-ATPase inhibitor). In the present study, thapsigargin in Ca2+-free Locke's solution, but not ryanodine in this solution, was found to prevent AAT degradation. AAT degradation was also prevented by calpain inhibitors (Ca2+-dependent protease inhibitor) such as calpeptin at 1 nM, 10 nM, 0.1 microM, 1 microM and 10 microM, and by calpain inhibitor peptide, but not by other protease inhibitors (10 microM leupeptin, pepstatin, chymostatin). Additionally, we determined the subcellular localization of calpain activity and examined the change of calpain activity in ischemic rat retinas. Our results suggest that decreased activity of mAAT in ischemic and hypoxic rat retinas might be evoked by the degradation by calpain-catalyzed proteolysis in mitochondria.