Molecular abnormalities of coproporphyrinogen oxidase in patients with hereditary coproporphyria

Genetic defects of coproporphyrinogen oxidase (CPO) lead to hereditary coproporphyria, an inherited autosomal dominant porphyria. The recent cloning of human cDNAs and of the gene encoding CPO permits deducing the primary structure of the CPO protein and elucidating the molecular basis of HC in some families.     

Decreased lymphocyte coproporphyrinogen III oxidase activity in hereditary coproporphyria

Using [${}^{14}\text{C}$]Coproporphyrinogen obtained from human red blood cells incubated with [$\text{4}{}^{14}\text{C}$] δ aminolevulinic acid, we measured lymphocytes Coproporphyrinogen III Oxidase activity in 17 subjects with hereditary coproporphyria. The mean activity was about 50 % of that in lymphocytes from normal subjects. This finding suggests that decreased coproporphyrinogen III oxidase activity reflects the primary genetic defect in Hereditary Coproporphyria. The technique described allows easy detection of asymptomatic carriers.     

A novel missense mutation in exon 4 of the human coproporphyrinogen oxidase gene in two patients with hereditary coproporphyria

Hereditary coproporphyria (HCP) is an autosomal dominant disease characterized by a deficiency of coproporphyrinogen oxidase. To date, four mutations of the gene have been reported. We report here another mutation in two Japanese families with HCP, which was revealed by analysis of polymerase chain reaction (PCR)-amplified DNA fragments of the gene by a direct-sequencing method. A point mutation, G to A, was found in exon 4 of the gene at position 538 of the cDNA from the reported putative translation initiation codon ATG. This mutation results in a glycine to arginine substitution at amino acid 180. Two carriers in the family were successfully diagnosed by detecting the mutation using restriction analysis of the PCR products. Received: 23 April 1996 / Revised: 15 July 1996     

Glucose phosphate isomerase deficiency with hereditary hemolytic anemia in a Spanish family: clinical and familial studies.

A new case of glucose phosphate isomerase deficiency associated with cogenital nonspherocytic hemolytic anemia is described in a 12-year-old girl of Spanish origin. The parents exhibited erythrocyte glucose phosphate isomerase activity between 50 and 60% of normal. The enzyme of the propositus had normal Michaelis-Menten constants both for F-6-P and G-6-P, but abnormal pH optimum and decreased heat stability at 48 degrees C. On starch-gel electrophoresis the father's enzyme was normal but the mother's showed a cathodic migrating band in addition to the normal one. The enzyme from the propositus exhibited only one band with cathodal mobility of 116% of the main band found in normal subjects. It is postulated that the propositus is double heterozygous for two abnormal alleles, and the mother contributes a mutant allele with abnormal electrophoretic mobility and thermolability at 48 degrees C whereas the father contributes an allele without enzymatic activity.     

A deficiency of coproporphyrinogen III oxidase causes lesion formation in Arabidopsis

We isolated an Arabidopsis lesion initiation 2 (lin2) mutant, which develops lesion formation on leaves and siliques in a developmentally regulated and light-dependent manner. The phenotype of the lin2 plants resulted from a single nuclear recessive mutation, and LIN2 was isolated by a T-DNA tagging approach. LIN2 encodes coproporphyrinogen III oxidase, a key enzyme in the biosynthetic pathway of chlorophyll and heme, a tetrapyrrole pathway, in Arabidopsis. The lin2 plants express cytological and molecular markers associated with the defense responses, usually activated by pathogen infection. These results demonstrate that a porphyrin pathway impairment is responsible for the lesion initiation phenotype, which leads to the activation of defense responses, in Arabidopsis. Lesion formation was not suppressed, and was even enhanced when accumulation of salicylic acid (SA) was prevented in lin2 plants by the expression of an SA-degrading salicylate hydroxylase (nahG) gene. This suggests that the lesion formation triggered in lin2 plants is determined prior to or independently of the accumulation of SA but that the accumulation is required to limit the spread of lesions in lin2 plants.     

3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals

ABSTRACT: BACKGROUND: Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the alpha and beta subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults. METHODS: We report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby. RESULTS: Fifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date. CONCLUSIONS: Our data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course.     

Lysyl oxidase deficiency in lung and fibroblasts from mice with hereditary emphysema.

Lysyl oxidase activity was measured in the lungs and from cultured fibroblasts of Blotchy mice. A marked decrease in lysyl oxidase activity was observed in lungs of affected mice as compared to normal litter mates. Fibroblasts cultured from Blotchy mice were also deficient in lysyl oxidase, producing less than half of normal enzyme levels. Normal and Blotchy fibroblasts which had been maintained in culture for several months and had undergone spontaneous transformation, continued to show the same magnitude of difference in lysyl oxidase levels. The data suggest that the deficiency of lysyl oxidase is inherent in Blotchy fibroblasts and support the idea that the deficiency of this enzyme is the metabolic lesion that leads to the connective tissue defects observed in these animals.     

Molecular characterisation of coproporphyrinogen oxidase from Glycine max and Arabidopsis thaliana

Coproporphyrinogen III oxidase (CPO; E.C. 1.3.3.3 ) is an enzyme of haem and chlorophyll synthesis. Biochemical studies have indicated that the majority of CPO activity is present in plastids, with no detectable levels in mitochondria. However, this approach cannot rule out low (less than 5%) activity in the mitochondria, nor the possible presence of CPO in the cytosol, where it is found in yeast (Saccharomyces cerevisiae). We have studied this question further using molecular techniques. A cDNA encoding the mature protein of soybean (Glycine max L.) CPO was used to overexpress the enzyme 200-fold in Escherichia coli. The recombinant enzyme, purified to homogeneity in three steps, is a dimer, with a K_m for coproporphyrinogen III of 0.25 ± 0.03 μM and a V_max of 1.48 pkat. Antibodies raised against the purified soybean CPO were used in western blots to show that the enzyme is present in etioplasts but not in mitochondria. In the completely sequenced genome of Arabidopsis thaliana, we identified two genes encoding CPO, but only one of them (AtCPO-I ) was able to complement a yeast mutant defective in the enzyme; the other is likely to be a pseudogene. A construct encoding the first 92 residues of AtCPO-I fused to green fluorescent protein (GFP) was introduced into Arabidopsis plants by Agrobacterium-mediated transformation. Confocal microscopy demonstrated that the CPO–GFP fusion protein was confined exclusively to plastids in leaves and roots, with no GFP seen in the mitochondria or cytosol.     

Molecular, biochemical and immunological analyses of porcine pancreatic DNase I.

Deoxyribonuclease I (DNase I) was purified 26500-fold in 39% yield from porcine pancreas to electrophoretic homogeneity using three-step column chromatography. The purified enzyme was inhibited by an antibody specific to the purified enzyme but not by G-actin. A 1303 bp cDNA encoding porcine DNase I was constructed from total RNA from porcine small intestine using a rapid amplification of cDNA ends method, followed by sequencing. Mature porcine DNase I protein was found to consist of 262 amino acids. Unlike all other mammalian DNase I enzymes that are inhibited by G-actin, porcine DNase I has H65 and S114 instead of Y65 and A114, which presumably results in the lack of inhibition. Porcine DNase I was more sensitive to low pH than rat or bovine enzymes. Compared with their primary structures, the amino acid at position 110 was N in porcine enzyme, but S in rat and bovine enzymes. A porcine mutant enzyme in which N was substituted by S alone at position 110 (N110S) became resistant to low pH to a similar extent as the rat and bovine enzymes.     

Genetic and biochemical impairment of mitochondrial complex I activity in a family with Leber hereditary optic neuropathy and hereditary spastic dystonia.

A rare form of Leber hereditary optic neuropathy (LHON) that is associated with hereditary spastic dystonia has been studied in a large Dutch family. Neuropathy and ophthalmological lesions were present together in some family members, whereas only one type of abnormality was found in others. mtDNA mutations previously reported in LHON were not present. Sequence analysis of the protein-coding mitochondrial genes revealed two previously unreported mtDNA mutations. A heteroplasmic A-->G transition at nucleotide position 11696 in the ND4 gene resulted in the substitution of an isoleucine for valine at amino acid position 312. A second mutation, a homoplasmic T-->A transition at nucleotide position 14596 in the ND6 gene, resulted in the substitution of a methionine for the isoleucine at amino acid residue 26. Biochemical analysis of a muscle biopsy revealed a severe complex I deficiency, providing a link between these unique mtDNA mutations and this rare, complex phenotype including Leber optic neuropathy.     

Molecular and biochemical characterization of a cytokinin oxidase from maize

It is generally accepted that cytokinin oxidases, which oxidatively remove cytokinin side chains to produce adenine and the corresponding isopentenyl aldehyde, play a major role in regulating cytokinin levels in planta. Partially purified fractions of cytokinin oxidase from various species have been studied for many years, but have yet to clearly reveal the properties of the enzyme or to define its biological significance. Details of the genomic organization of the recently isolated maize (Zea mays) cytokinin oxidase gene (ckx1) and some of its Arabidopsis homologs are now presented. Expression of an intronless ckx1 in Pichia pastoris allowed production of large amounts of recombinant cytokinin oxidase and facilitated detailed kinetic and cofactor analysis and comparison with the native enzyme. The enzyme is a flavoprotein containing covalently bound flavin adenine dinucleotide, but no detectable heavy metals. Expression of the oxidase in maize tissues is described.     

Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.     

Inositol deficiency in yeast: metabolic,enzymatic and autoradiographic studies

The addition of inositol to starved cells of Saccharomyces cerevisiae NCYC 86 resulted in an initiation of growth. Inositol was incorporated into phosphatidylinositol and after a lag period RNA was the first macromolecule with a rate of synthesis departing from the rate observed in deprived cells. Pulse chase experiments showed that inositol was first incorporated into phosphatidylinositol and later into more polar lipids. Finally it appeared to be excreted into the surrounding medium. When S. cerevisiae NCYC 86 was grown in suboptimal concentrations of inositol (0,5 g/ml), alterations in the level of some membrane-bound enzymatic activities were detected; these might reflect structural modifications of the cellular membranes due to a different composition of phospholipids.High-resolution autoradiography showed that inositol was probably first incorporated into internal membranes and later transferred to the plasma membrane. Analytical experiments carried out with inositol-deprived cells showed that inositol was released into the surrounding medium in that case.The unbalanced growth detected in S. cerevisiae NCYC 86 under inositol deprivation might be due to an abnormal functioning of the cell membranes as a consequence of the deficiency in inositol-containing phospholipids.     

Molecular analysis of a novel hereditary C3 deficiency with systemic lupus erythematosus

A case of inherited homozygous complement C3 deficiency (C3D) in a patient with systemic lupus erythematosus (SLE) and the molecular basis for this deficiency are reported. A 22-year-old Japanese male was diagnosed as having SLE and his medical history revealed recurrent tonsillitis and pneumonia. He was diagnosed as having C3D because of undetectable serum C3 level. His parents were consanguineous. Sequence analysis of C3D cDNA revealed a homozygous deletion of exon 39 (84bp). A single base substitution (AG to GG) in the 3'-splice acceptor site of intron 38 was identified by sequencing the genomic DNA. Expression of C3Delta(ex39) cDNA, the C3cDNA lacking exon 39, in COS-7 cells revealed that C3Delta(ex39) was retained in endoplasmic reticulum-Golgi intermediate compartment because of defective secretion. These data indicate that a novel AG-->GG 3'-splice acceptor site mutation in intron 38 caused aberrant splicing of exon 39, resulting in defective secretion of C3.