Erythrocyte Cytochrome B5 Research Articles

Identification of Two Novel Mutations in the CYB5R3 Gene in Five Cases of Type I Methemoglobinemia

Congenital methemoglobinemia due to cytochrome b5 reductase (CYB5R3) deficiency are sporadic worldwide, although clusters of this disorder have been identified in certain ethnic groups. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients representing three distinct ethnic backgrounds. Three subjects were of Indian ethnicity one being phenotypically normal and his two cousins diagnosed with type I methemoglobinemia. Causative mutations in the globin genes were excluded after sequencing. A fourth subject, a 12 year old Mexican child with Turner's syndrome and apparent congenital cyanosis was referred to us for evaluation. Her methemoglobin level was elevated and erythrocyte cytochrome b5 reductase was reduced (4% of the normal range). The fifth individual, a 36 year old woman of Greek ancestry, had a past history of childhood cyanosis and low O2 saturation (82%) post-surgery in 2007. In early 2008 she was diagnosed with methemoglobinemia due to dyspnea, disproportionate fatigue, and significant increase in methemoglobin levels (10%). Genomic DNA from all 5 subjects was isolated, and all nine exons of the CYB5R3 gene were PCR amplified and sequenced. A non-synonymous heterozygous mutation (G>A transversion) in exon 6 resulting in the change of amino acid 178 from alanine to threonine (A178T) was identified in both affected Indian siblings and not in their unaffected cousin. This mutation which is located in the NADH binding domain and in close proximity to the adenosine moiety, and may influence binding of the NADH coenzyme had been previously reported (Dekker et al., 2001). A novel homozygous mutation in an evolutionarily conserved position located in exon 3 (C>G transition) resulting in substitution of serine 53 for arginine (S53R) was found in the child of Mexican descent. This mutation, located within the FAD binding domain, is hypothesized to cause disruption of hydrogen bonding and steric hindrance due to the bulkier arginine side chain, consequently leading to protein instability. Five heterozygous mutations were detected in the subject with Greek ancestry. A synonymous G>A substitution (Pro44Pro) in exon 2, a previously reported type II mutation G>A (Val253Met) and two 3′-UTR single nucleotide polymorphisms (A>G at 29690 nt, and C>A at 29914 nt), all in exon 9, were found in a cis configuration. A novel heterozygous compound mutation in exon 6, a T>G transversion, resulting in substitution of phenylalanine 157 for cysteine (F>C) was identified in the same subject with Greek ancestry (Figure 1). PCR amplified CYB5R3 transcripts harboring either wild-type, S53R, or F157C sequences were cloned, in frame with the 10X histidine tag, into the pET16b expression vector. Sequence fidelity and in frame fusion of all constructs was verified by sequencing from both strands. Recombinant protein was purified using 1 mL niquel-affinity columns. Kinetic studies using recombinant CyB5R3 enzyme will follow. [Display omitted]

Stage-specific isozymes of ascaris suum complex II: the fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae have the common iron-sulfur subunit

The cytochrome b5 of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a novel type of cytochrome b5. It is a soluble protein that lacks the COOH-terminal membrane-anchoring domain found in erythrocyte cytochrome b5, but possesses an NH2-terminal extension (presequence) of 30 amino acids that are missing from the 82-residue protein purified from the nematode tissues [Yu, Y., Yamasaki, H., Kita, K., and Takamiya, S., 1996, Arch. Biochem. Biophys. 328, 165–172]. The nematode cytochrome b5 is, therefore, probably synthesized as a precursor protein whose presequence is cleaved to form a mature protein, but the localization of the mature protein is still unknown. To investigate the processing of the putative precursor protein, the wild-type precursor of nematode cytochrome b5 with a complete presequence (b5wt) and its NH2 terminus-truncated derivatives, b5Δ18 and b5Δ28, with 18 and 28 residues deleted, respectively, were expressed using pET-28a(+) vector in Escherichia coli. As expected, all transformants, tb5wt, tb5Δ18, and tb5Δ28, produced recombinant proteins with a histidine-tagged NH2-terminal extension. However, only the recombinant protein with the full-length presequence, produced in tb5wt, was correctly processed and transported to the periplasm, from which the majority of the induced product was purified as a mature protein chemically and functionally identical to the native protein purified from the nematode body wall. These results clearly show that the nematode histidine-tagged presequence functions as a signal peptide in E. coli.

Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants

SummaryMessenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector λgt11 with an efficiency of cloning of approximately 104 recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the β-galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.

Improved determination of cytochrome b5 in human erythrocytes.

A sensitive, precise enzymic/spectrophotometric method for determining cytochrome b5 in small amounts of blood is described. Mean values for healthy individuals, ages 20 to 70 years, were 0.26 (SD 0.03) mumol per liter of erythrocytes or 0.87 (SD 0.14) nmol per gram of hemoglobin. We believe the assay is preferable to methods described hitherto, primarily because of its high sensitivity.

Catalysis of Methemoglobin Reduction

This chapter focuses on the catalysis of methemoglobin reduction. It discusses the results of studies in which photometry and magnetic circular dichroism (MCD) spectroscopy were used to follow the kinetics of methemoglobin reduction. The chapter discusses an experimental study in which methemoglobin reduction in hemolysates was assayed. The hemolysate differs from conditions within the normal erythrocytes in that nitrite was added to convert hemoglobin to methemoglobin. The components of the cytoplasm were diluted by a factor of 4.8, the membranous fraction was removed, and NADH or NADPH was added to a concentration of 4 mM. In this system, the dependence of rate on the concentrations of the various components was studied by addition to the hemolysate of purified human methemoglobin, human erythrocyte cytochrome b5reductase, or bovine erythrocyte cytochrome b5. Direct reduction of methemoglobin by the addition of isolated ferrocytochrome b5was measured photometrically. The reaction was carried out at 37°C in a cuvette, which was already positioned in an Aminco recording spectrophotometer. Photochemical reduction mediated by lumiflavin 3-acetate was employed to reductively titrate mixtures of cytochrome b5and methemoglobin. MCD spectra were obtained with a Jasco J-40C spectropolarimeter equipped with an electromagnet. Spectra were acquired, stored, corrected, and analyzed by using a Nova III computer, a Tracor 1500N signal analyzer, an Ohio Scientific Challenger II microcomputer, and an ADM 3a terminal retrofitted with Retro-Graphics plotting hardware.

Purification and structural studies of rabbit erythrocyte cytochrome b5

A single form of cytochrome b5 has been isolated in highly purified form from the cytosolic fraction of rabbit erythrocytes by sequential chromatography on DE-52 cellulose, Sephadex G-75, and DEAE-Sephadex A50. The cytochrome is structurally similar to the N-terminal, heme-binding domain of rabbit liver microsomal cytochrome b5. Like the liver protein, it is blocked at the amino terminus. Its amino acid composition is similar to that of residues 1-97 of the microsomal protein. With one exception, tryptic peptides derived from apo-cytochrome b5 of rabbit erythrocytes co-elute with the tryptic peptides obtained from a soluble hemepeptide fragment of microsomal cytochrome b5. These findings, together with amino acid sequence analysis of the carboxyl terminal tryptic peptides, identify the erythrocyte cytochrome b5 as a 97-residue peptide.

Demonstration that bovine erythrocyte cytochrome b5 is the hydrophilic segment of liver microsomal cytochrome b5

A structural comparison has been made between bovine erythrocyte cytochrome b 5 and solubilized forms of bovine hepatic microsomal cytochrome b 5. Two soluble forms of microsomal cytochrome b 5 (designated Forms A and B) were generated by digestion of microsomes with a crude hepatic lysosomal cathepsin preparation and purified by successive chromatography on DEAE-cellulose,Bio-Gel P-60 and DEAE-Sephadex A-50.Amino acid analyses and terminal residue analyses identified Form A as the segment corresponding to residues 1∗95 of the native microsomal protein and Form B as the segment corresponding to residues 1∗107. Erythrocyte cytochrome b 5 I was shown to be a protein which corresponds to a segment of the hepatic microsomal molecule containing residues 1∗97, whereas erythrocyte cytochrome b 5 II is a protein corresponding to residues 1∗95. Like the native microsomal cytochrome and the cathepsin-solubilized forms of the cytochrome, no amino terminal residue could be detected in the erythrocyte cytochrome.Carboxypeptidases A and B released from erythrocyte Form I a residue eluting at the position of serine, but released no residue from Form II. The results are consistent with serine being the residue at position 97 of the native microsomal protein, and proline and serine being the residues in positions 94 and 95, respectively. The maps of the tryptic peptides derived from the apoprotein forms of erythrocyte cytochrome b 5 I and II and cathepsin-solubilized microsomal Forms A and B were very similar, with eight of the expected twelve peptides displaying the same mobility on every map. Amino acid analyses of the isolated tryptic peptides from erythrocyte Form I and hepatic Form B confirmed the structural assignments of these proteins. These data demonstrate that the soluble forms of erythrocyte cytochrome b 5 correspond to hydrophilic segments of the native membrane-bound microsomal cytochrome b 5 and suggest that the hepatic lysosomal proteases serve as a good model for the putative erythroid proteases which solubilize microsomal cytochrome b 5 during erythroid maturation.

NADH cytochrome b5 reductase activity in lymphoid cell lines. Expression of the defect in epstein Barr virus transformed lymphoblastoid cell lines from patients with recessive congenital methemoglobinemia.

Recessive congenital methemoglobinemia (RCM) is due to the homozygous deficiency of NADH-cytochrome b5 reductase (EC 1.6.2.2.). In type I disease, in which the patients are only methemoglobinemic, the enzyme defect is fully expressed in the erythrocytes, whereas the leukocytes are much less affected. In type II disease, in which the patients are, in addition, mentally retarded, the defect is generalized to all the tissues including cultured fibroblasts. In the present study we have investigated Epstein-Barr virus (EBV) transformed lymphoid cell lines (LCL) derived from patients with both types of cytochrome b5 reductase deficiency and from nondeficient individuals. The total cytochrome b5 reductase activity of the control LCL was found to be similar whatever the LCL origin, except for one lymphoma line (Daudi). The enzyme from the control LCL (c 252/B 95) was found to be immunologically related to the human soluble erythrocyte cytochrome b5 reductase, indicating that it is the product of the same gene: the DIA1 (diaphorase) locus. The LCL derived from one patient with the type I disease and two patients with the type II disease were investigated.l In the former the defect was expressed to a lesser degree than in the cases with mental retardation in which the defect was much pronounced, and involved both the mitochondrial and the microsomal fraction. This indicated that all the subcellular forms of the cytochrome b5 reductase are under the same genetic control. Altogether, these data show that the LCL are a favorable material for studying both types of cytochrome b5 reductase deficiency and for investigating in depth the molecular aspects of this metabolic disease.

Properties of methemoglobin reductase and kinetic study of methemoglobin reduction.

A soluble erythrocyte cytochrome b5 was purified as the substrate of methemoglobin reductase and an electron carrier to methemoglobin. The isoelectric point of this protein was at pH 4.3, and E0' was -0.010 at pH 7.0.. The Km value of the enzyme for this protein was 1 x 10(-4) M, and the turnover number (k5) was 3.4 x 10(4) min-1, with NADH as an electron donor at pH 7.0. The optimum pH of the enzyme was pH 4.6 for ferricyanide and pH 5.5 for cytochrome b5, with a shoulder of activity at pH 7 to 9 for both substrates. The rate equation which represents the reduction of either methemoglobin or cytochrome c was obtained as a function of methemoglobin or cytochrome c, methemoglobin reductase, and cytochrome b5 by considering the E . S complex for both reductase and cytochrome b5, and the rate constants involved were determined. The rate constants between methemoglobin and reduced cytochrome b5 (k1, M-1 min-1) were 1.6 x 10(4), 3.1 x 10(6), and 4.1 x 10(6) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constants between the reduced enzyme and oxidized cytochrome b5 (k'3, M-1 min-1) were 4.3 x 10(8), 12 x 10(8), and 9.3 x 10(8) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constant between reduced hemoglobin and oxidized cytochrome b5 (k2) was 35 M-1 min-1 at pH 7.0. The theoretical Km for methemoglobin was 2.1 M at an infinite enzyme concentration at pH 7.0

Age-dependent decay of cytochrome b5 and cytochrome b5 reductase in human erythrocytes

Age-dependent decrease in cytochrome b5 was observed in erythrocytes from both a normal person and a patient with hereditary methaemoglobinaemia without neurological symptoms. With aging, concentrations of cytochrome b5 in erythrocytes from the patient were almost the same as those in the control. Age-dependent decrease in cytochrome b5 reductase activity in the control erythrocytes was also shown; however, the reductase activity was very low in erythrocytes from the patient over the whole age range. Our studies show that methaemoglobin content of erythrocytes seems to be dependent on the content of cytochrome b5 in the cells, both in the control subject and in the patient.

A simple enzymatic microdetermination of cytochrome b5 in erythrocytes

A simple enzymic method for the microscale determination of erythrocyte cytochrome b 5 is presented. For assay of cytochrome b 5 the sodium nitrite-treated hemolysate was incubated in the presence of NADH and NADH-cytochrome b 5 reductase, and the reduction of methemoglobin was measured. The rate of methemoglobin reduction was linearly dependent on the amount of cytochrome b 5. When the red cells were stored at 4°C or methemoglobin-formed hemolysates were stored at 4°C or −20°C, there was no significant decline in the amount of cytochrome b 5 for periods of at least 2 weeks. Values for normal human subjects showed a mean of 0.70 ± 0.17 SE nmol/ml of red cells for males, 0.52 ± 0.11 nmol/ml red cells for females, and 0.48 ± 0.15 nmol/ml red cells for umbilical cord bloods.

Properties of cytochrome b5 and methemoglobin reduction in human erythrocytes.

Cytochrome b5 of human erythrocytes showed a midpoint redox potential of - 2 mV, an isoelectric point of 4.3, and a Michaelis constant of 7μ for erythrocyte cytochrome b5 reductase These values are close to those of rabbit liver cytochrome b5, though cytochrome b5 of erythrocytes is in the soluble fraction and its relative molecular mass is smaller than that of liver cytochrome b5 The concentration of cytochrome b5 in normal human erythrocytes was determined by an improved method and a value of 0.22 ± 0.02 μM was obtained. The optimum pH of the cytochrome b5 reduction by the reductase from human erythrocytes was 5.5, and about half of the maximum activity was observed at neutral pH. As electron donors, α-NADH and NADPH were 56% and 1 % as effective as β-NADH respectively. A Michaelis constant of 22 nM for β-NADH was obtained by fluorometric assay method. The rate of cytochrome b5 reduction in normal human erythrocytes was calculated to be 1.4 mM/h by the use of the substrate concentrations in vivo. The rate constant of the nonenzymatic methemoglobin reduction by reduced cytochrome b5 was determined to be 6.2 × 103 M-1 s-1. These values account for the methemoglobin reduction rate in human erythrocytes on the hypothesis proposed previously (Hultquist, Sugita), namely methemoglobin is reduced nonenzymatically by cytochrome b5 which has been reduced by NADH-cytochrome b5 reductase.

Evidence that two forms of bovine erythrocyte cytochrome b5 are identical to segments of microsomal cytochrome b5.

Homogeneous preparations of two forms of soluble cytochrome b5 have been obtained from bovine erythrocytes by successive chromatography on DEAE-cellulose, Bio-Gel P-60, and DEAE-Sephadex. Although the two forms could be separated on disc gel electrophoresis, they appeared to have similar molecular weights of approximately 12,000 and identical visible absorbance spectra. The tryptic hemepeptides derived from the two forms of bovine erythrocyte cytochrome b5 are electrophoretically indistinguishable from each other and from the tryptic core hemepeptide derived from liver microsomal cytochrome b5. The bovine erythrocyte tryptic hemepeptide was purified to homogeneity; its amino acid composition was shown to be identical to that of tryptic hemepeptide from liver microsomal cytochrome b5. The amino acid compositions of the two isolatable forms of erythrocyte cytochrome b5 correspond well to the compositions of the 97- and 95-residue segments of native liver microsomal cytochrome b5 that begin at the NH2 terminus. These results agree with the hypothesis that soluble erythrocyte cytochrome b5 is derived from microsomal protein by proteolysis during erythroid maturation.

Catalysis of methaemoglobin reduction by erythrocyte cytochrome B5 and cytochrome B5 reductase.

THE reduction of methaemoglobin is one of the few oxidation-reduction reactions known to occur in the mature red blood cell. Approximately 3% of the circulating haemoglobin of an adult man is oxidized each day to methaemoglobin1 and so a methaemoglobin reduction system is needed in the cell to maintain a low concentration of methaemoglobin (normally less than 1% of the total haemoglobin2).