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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3250-3255
RED CELLS
From Research Laboratories, Center for Laboratory Medicine, Fuzhou
General Hospital, Fuzhou City, Fujian Province, China; and Zhousan
People's Hospital, Zhousan City, Zhejiang Province, China.
Recessive congenital methemoglobinemia due to nicotinamide adenine
dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is
classified into 2 clinical types: type 1 (erythrocyte type) and type 2 (generalized type). We found a Chinese family with type 1 recessive
congenital methemoglobinemia, the patients from which were diagnosed
according to clinical symptoms and b5R enzyme activity in the blood
cells. To learn the molecular basis of type 1 recessive congenital
methemoglobinemia in this Chinese family, we isolated total RNA from
the peripheral leukocytes of the propositus and b5R complementary
DNA (cDNA) by reverse transcription- polymerase chain reaction
(RT-PCR). The coding region of the b5R cDNA was analyzed by sequencing
the cloned PCR products. The results showed that the propositus was
homozygous for a G
Nicotinamide adenine dinucleotide (NADH)-cytochrome b5
reductase (b5R, EC.1.6.2.2.) deficiency leads to 2 different types of
recessive congenital methemoglobinemia. In type 1, cyanosis is the only
major symptom, and b5R deficiency is restricted to red blood
cells.1 In type 2,2 cyanosis is associated with severe mental retardation and neurologic impairment, and the enzyme deficiency is systemic. The b5R gene is a housekeeping gene. A membrane-bound form of b5R is found in all somatic cells and plays an
important role in fatty acid metabolism, cholesterol synthesis, and in
vivo transformation of drugs.3 Soluble-form b5R exists mainly in erythrocytes, where it functions in the reduction of methemoglobin. It is of great interest to learn the molecular basis of
recessive congenital methemoglobinemia, because identification of
different mutations occurring at different positions within the b5R
gene might account for the phenotypic heterogeneity of this disease. In
previous reports we have described 2 point mutations (Arg57Gln and
Leu72Pro) in the b5R gene of Chinese families of type 1 recessive
congenital methemoglobinemia.4,5 In this paper we present
analysis of the b5R gene of another Chinese pedigree by reverse
transcription-polymerase chain reaction (RT-PCR), PCR-restriction fragment length polymorphism (RFLP), and dot blot hybridization, with
the identification of a G Case report and family pedigree
Electrophoresis and visualization of red cell b5R on native
polyacrylamide gel
Determination of b5R protein in the erythrocytes by
double-antibody sandwich enzyme-linked immunoabsorbent assay
RNA isolation and RT-PCR Total RNA was extracted from peripheral lymphocytes of the propositus and an unrelated normal control by using RNeasy Blood Mini Kit (Qiagen, Hilden, Germany). The synthesis of complementary DNA (cDNA) was performed from 1 µg of total RNA according to the instructions of the kit producer (Promega, Madison, WI). The entire b5R cDNA coding region starting with the ATG initiation site in exon 1 through the TGA stop site in exon 9 was amplified with 1 primer set (P1: 5'-GGGAATTCATGGGGGCCCAGCTCAGCACG-3'; P2: 5'-GGGGATCCCCTCAGAAGACGAAGCAGCGC-3'; synthesized on an Applied Biosystems DNA synthesizer [Perkin Elmer, Foster City, CA]) that allows the amplification of a 921-base pair (bp) fragment. The amplification reaction was performed on Gene Amp system 2400 (Perkin-Elmer Setups ) in a 50 µL reaction volume containing 5 µL of 10 × PCR buffer (500 mmol/Lol/L KCl; 100 mmol/L Tris-HCl, pH 9.0; 1% Nonodet P40), 5 µL of 25 mmol/L MgCl2, 2U of Taq polymerase (Sangon, Shangai, China), 5 µL of 2 mmol/L of each dNTP, 1 µL of 10 pmol/L of each primer, and 4 µL of cDNA. PCR reaction was started with an initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 45 seconds, annealing at 60°C for 45 seconds, and elongation at 72°C for 60 seconds, with an additional extension at 72°C for 10 minutes after the last cycle. The PCR products were visualized by electrophoresis on 1.0% agarose gel stained with ethidium bromide.Cloning and nucleotide sequencing of the amplified products The RT-PCR products were purified from the agarose slice after gel electrophoresis by phenol-chloroform extraction. The purified PCR fragments were cloned into the vector pWR-4509 (kindly provided by Prof Xie Yi of Fudan University, Shanghai, China) by overnight ligation at 12°C using T4 DNA ligase (MBI Fermentas, Milan, Italy). The recombinant vector was used to transform Escherichia coli JM103. Clones of interest were screened by PCR and sequenced with the P1 and P2 primers on ABI 373A sequencer by following a dye terminator protocol.Genomic DNA amplification and PCR/restriction analysis The peripheral lymphocytes of the propositus, her family members, and an unrelated normal control were washed twice with phosphate-buffered saline, and genomic DNA was extracted using QiAamp Blood Kit (Qiagen). The genomic DNA was amplified with a primer set10 (5'-AGTACACCTGGGCGGGGCGG-3' and 5'-AGAATCTAGCAGAGCTGTGCA-3') that allows the amplification of a 262-bp fragment of exon 7. The PCR reaction volume was 25 µL, containing 2.5 µL of 10 × PCR buffer, 2.5 µL of 25 mmol/L MgCl2, 2.5 µL of 2 mmol/L of each dNTP, 1 µL of 10-mmol/L of each primer, and 2U of Taq polymerase (Sangon). Thirty cycles of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and elongation at 72°C for 1 minute were carried out, followed by an extended incubation at 72°C for 10 minutes. The amplified products were purified by phenol-chloroform extraction, digested with RsaI (MBI) at 37°C for 60 minutes, and subjected to electrophoresis in 12% polyacrylamide gel.Dot blot hybridization with allele-specific oligonucleotide probes Digoxigenin (DIG) Oligonucleotide Tailing Kit and DIG Nucleic Acid Detection Kit were purchased from Boehringer Mannheim (Mannheim, Germany). PCR products of genomic DNA from the propositus, her family members, and 3 unrelated normal controls were loaded onto nitrocellulose membranes and then baked for 2 hours at 80°C. The 1-hour prehybridization was done at 54°C for the mutant probe or at 60°C for the normal probe in prehybridization buffer: 5 × SSC (0.75 mol/L NaCl, 0.075 mol/L sodium citrate, pH 7.0), 1% blocking reagent (in 0.1 mol/L maleic acid, 0.15 mol/L NaCl, pH 7.5), 0.1% N-lauroylsarcosine, 0.02% SDS. The probe WA I (5'AGAGCAGGTGGCACACAG-3') for normal allele (normal probe) and the probe WA II (5'-ACTGTGTACCACCTGCTCT-3') for the mutated allele (mutant probe) were labeled by tailing incorporation of a digoxigenin-labeled nucleotide. The hybridization reaction was initiated by adding the labeled probes directly to the prehybridization mixture. After 6 hours of hybridization, the membranes were washed twice for 5 minutes with 2 × SSC, 0.1% SDS at room temperature, and twice for 15 minutes with 0.1 × SSC, 0.1% SDS at 60°C for the normal probe or at 54°C for the mutant probe. The immunologic detection was carried out as described in the product manual. Briefly, the filters were incubated at room temperature for 30 minutes in 1% blocking reagent containing 150 mU/mL anti-digoxigenin-alkaline phosphatase conjugate. The color reaction was initiated by the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt and was completed in 16 hours.Expression, purification, and characterization of GST-fused b5R To construct glutathione S-transferase (GST)-fused b5R expression vector, cDNA fragments encoding soluble-form normal and mutant b5R were isolated by PCR from the corresponding pWR-450 recombinants with 1 primer set (5'-GGGAATTCCCCTCAGAAGACGAAGCA-3' and 5'-GGGGATCCTTCCAGCGCTCCACGCC-3'). The wild-type and mutant b5R cDNA fragments were inserted into the GST fusion gene vector, pGEX-2T (Pharmacia, Piscataway, NJ). Only 1 extra amino acid residue was introduced into the cloning (BamHI) site. E. coli BL21 harboring the expression plasmid was incubated overnight at 37°C in 3 mL of L-broth medium containing 100 µg/mL ampicillin. The culture was inoculated into 50 mL of L-broth medium and incubated at 37°C. After an initial incubation for 2.5 hours, isopropyl- -D-thiogalactopyranoside was added to the medium, with a
final concentration of 0.1 mmol/L, and incubated for another 4 hours.
The bacterial cells were pelleted by centrifugation (5000g for
5 minutes), resuspended in sterile water, lysed by sonication, and spun
at full speed on a microcentrifuge for 10 minutes to remove insoluble
materials. The supernatant was transferred to Glutathione Sepharose 4B
Microspin Column (Pharmacia) and mixed gently at room temperature for
10 minutes to ensure optimal binding of GST proteins to the Glutathione
Sepharose 4B matrix. After washing the column twice with
phosphate-buffered saline, elution was performed with the elution
buffer containing 10 mmol/L reduced glutathione. The concentrations of
fusion proteins were determined by measuring light absorbance at 280 nm. Purity of the GST-fused b5Rs was evaluated by 10%
SDS-polyacrylamide gel electrophoresis.6 Western blot
analysis of GST-fused b5Rs was performed according to the standard
method.11 GST-fused b5Rs were separated on 10%
SDS-polyacrylamide gels and blotted onto nitrocellulose filters by
eletrophoretic transfer. The nitrocellulose filters were incubated with
anti-b5R monoclonal antibody (1E3)12 for 1 hour at room
temperature. Biotinylated goat antimouse immunoglobulin G was used as
the second antibody (Zymed, San Francisco, CA). The GST-fused b5Rs were
identified on the membrane by color development using
4-chloro-1-naphthol and H2O2 as the substrates
of streptavidin-conjugated horseradish peroxidase. A b5R preparation
(rb5R, kindly provided by Dr Yubisui, Oita University,
Oita, Japan) was used as control.
Analysis of enzyme properties of GST-fused b5Rs Kinetic parameters of the GST-fused wild-type b5R and GST-fused Cys203Tyr mutant b5R were assayed with DCIP as the electron acceptor.13 A standard reaction mixture (3 mL) contained 0.03 mmol/L NADH, 0.05 mmol/L Tris-HCl (pH 7.5), 20 µg of b5R fusion protein, 0.1 mmol/L ethylenediamine tetraacetic acid (EDTA), and 0.06 mmol/L DCIP. The Km values of each enzyme were determined by Lineweaver-Burk double-reciprocal plot. The Kcat values were calculated by dividing the maximal reaction velocity by the enzyme amount in mole. According to the method of Higasa et al,14 stability to heat was tested by incubating the b5Rs at various temperatures for 10 minutes and at 37°C for various times, and susceptibility to trypsin was examined by incubation for various times at 37°C with 1.5 U/µL trypsin. In such cases, the enzyme activity was checked by using potassium ferricyanide as an electron acceptor15 and expressed as percentages of the activity before heat or trypsin treatment for the sake of convenience. A standard reaction mixture contained 30 µL of 100 µmol/L NADH, 0.25 mL of 10 mmol/L EDTA, 0.5 mL of 50 mmol/L citrate buffer (pH 4.5), 0.8 mL of 0.2 mmol/L potassium ferricyanide, and 0.5 mL of 11.68 g/L b5R-free hemoglobin. NADH-ferricyanide activity was measured at 575 nm at 25°C with Shimadzu UV-240 spectrophotometer (Shimadzu, Kyoto, Japan).Other methods Total hemolysate hemoglobin concentrations were determined with an automatic blood analyzer (Coulter JT-IR, Miami, FL), the built-in protocol of which eliminates any interference of the determination by methemoglobin. Methemoglobin level was determined by the classical method of Evelyn and Malloy16 and expressed as a percentage of total hemoglobin (Table 1).
Visualization of b5R activity in native gel and quantification of b5R protein Red-cell b5R from the propositus, some of her family members, and a normal subject was studied by native gel electrophoresis. Submerging the gel in MTT-DCIP-NADH substrate solution to stain b5R in polyacrylamide gel. A diaphorase band appeared behind that of hemoglobin (Figure 2). No NADH-diaphorase activity can be observed in the hemolysates of the propositus and 2 of her brothers by this method. Her daughter and nephews had less intensive NADH-diaphorase bands than those of the normal subject. An ELISA method showed that the content of b5R protein in the erythrocytes of the daughter and nephews was much smaller than that of a normal control, and no b5R protein could be detected in the hemolysates of the propositus and 2 of her brothers (Table 1).
Sequence analysis of the patient's b5R gene A fragment of 921 bp from PCR amplification of b5R cDNA was detected on 1.0% agarose gel. This fragment was purified, cloned, and sequenced. Sequencing results (Figure 3) revealed only 1 notable base change in the propositus, a G A
transition in exon 7 at the second position of codon 203, causing an
amino acid change from cysteine to tyrosine.
PCR/restriction enzyme analysis and dot blot hybridization To confirm that this mutation was not an artifact derived from the misincorporation of Taq polymerase during PCR, we performed PCR-RFLP and dot blot hybridization. Because this nucleotide replacement created another RsaI recognition site within the 262-bp fragment (Figure 4A), the PCR products from genomic DNA were digested by RsaI and separated on 12% polyacrylamide gel. The 262-bp PCR product of the propositus was cleaved into 2 smaller fragments of 134 bp and 125 bp, as predicted (Figure 4B). In addition, the amplified DNA of the propositus, 5 of her family members, and 3 normal controls was allowed to interact with an oligonucleotide probe specific either for the normal (WA I) or for the mutant (WA II) allele. It was shown (Figure 5) that the mutant-specific probe was hybridized to the PCR products of the propositus and 2 of her brothers (II.3 and II.7) but not to any specimen from the normal individuals, and the normal probe was hybridized to the PCR products of the normal individuals but not to any specimen from the propositus and 2 of her brothers. The amplified DNA of her daughter (III.5) and her nephews (III.9 and III.10) could be hybridized to both the mutant and normal probes. These facts indicated that the propositus and her brothers were homozygous and her daughter and nephews were heterozygous for this GA substitution.
Preparation and identification of fusion-expressed b5R proteins To characterize the effect of the Cys203Tyr missense mutation on the biochemical properties of b5R, the normal and mutant b5R cDNAs were inserted into GST fusion gene expression vector separately. GST-fused wild-type b5R and GST-fused Cys203Tyr b5R were expressed in E. coli BL21and purified as described in "Materials and methods." A single band of 58 kd was observed after purification (Figure 6A). To determine whether GST-fusion proteins expressed in E. coli were b5R proteins, Western blot analysis was carried out with a monoclonal antibody against b5R. As shown in Figure 6B, a single band with molecular mass of 58 kd was revealed. These results confirmed that GST-fusion proteins expressed in E. coli were indeed b5R proteins.
Kinetic properties of the expressed enzymes The enzyme activity of mutant b5R was almost the same as that of the wild-type enzyme when measured at the same concentration. Kinetic properties of the wild-type and mutant enzymes are summarized in Table 2. The Km values of the GST-fused wild-type b5R for DCIP and NADH were 26 µmol/L and 68 µmol/L, respectively. The Km values of the mutant b5R were close to those of the wild type. Little difference between the wild-type and mutant b5Rs was noted in Kcat values, as well as in the Kcat/Km values. These results indicated that the kinetic properties of mutant enzyme b5R were essentially the same as those of the wild-type b5R, suggesting that the cysteine to tyrosine substitution at residue 203 does not affect the affinity for NADH and DCIP and the catalytic activity of b5R.
Heat stability and protease susceptibility of GST-fused b5Rs There were distinct differences in heat stability between the mutant enzyme and the wild-type enzyme. Residual activities of the mutant enzyme after 10 minutes at 37°C and 50°C were about 70% and less than 5% of the initial activity, respectively, whereas the wild-type b5R retained 50% of the initial activity after 10 minutes at 50°C (Figure 7A). After a 30-minute incubation at 37°C, enzyme activity of the wild type dropped only slightly (more than 95% initial activity), but mutant Cys203Tyr b5R was heat-inactivated to less than 70% of the initial activity under the same conditions (Figure 7B). We made use of trypsin to reveal the protease sensitivity of the mutant Cys203Tyr b5R. The mutant b5R showed only 5% of the initial activity after being incubated with 3 µmol/L trypsin for 30 minutes at 37°C, whereas the wild-type b5R showed much higher resistance to the action of trypsin, retaining 47% of the initial activity under the same conditions (Figure 7C).
Discussion The organization of the NADH-cytochrome b5 reductase gene has been determined in rats and humans. The human gene is about 31 kilobases long with 9 exons and 8 introns. The translational start site is in exon 1. Human liver b5R cDNA has a 903-bp open reading frame, predicting a protein of 301 residues.17 Identification of different mutations occurring at different positions within the b5R gene might account for the phenotypic heterogeneity of this disease. Meanwhile, the characterization of mutant b5R protein might provide insight into the understanding of the molecular basis of 2 types of recessive congenital methemoglobinemia and shed light on the correlation between protein structure and activity of mutant enzymes.
Acknowlegments We thank Mr Dezhu Zheng for determining hemoglobin concentration and Professor Yi Xie of Fudan University for providing pWR-450 plasmid and useful comments.
Submitted April 4, 1999; accepted January 6, 2000.
The sequence reported in this paper has been submitted to GenBank. The accession number is AJ0110118.
Reprints: Yao Wang, Research Laboratories, Center for Laboratory Medicine, Fuzhou General Hospital, 156 Xihuanbei Road, Fuzhou City, Fujian Province, 350025, People's Republic of China; e-mail: SZQ{at}public.fz.fj.cn or yctang{at}pub2.fz.fj.cn.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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  C. Ewenczyk, A. Leroux, A. Roubergue, V. Laugel, A. Afenjar, J. M. Saudubray, P. Beauvais, T. B. de Villemeur, M. Vidailhet, and E. Roze Recessive hereditary methaemoglobinaemia, type II: delineation of the clinical spectrum Brain, March 1, 2008; 131(3): 760 - 761. [Abstract] [Full Text] [PDF]
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J. Dekker, M. H. M. Eppink, R. van Zwieten, T. de Rijk, A. F. Remacha, L. K. Law, A. M. Li, K. L. Cheung, W. J. H. van Berkel, and D. Roos Seven new mutations in the nicotinamide adenine dinucleotide reduced-cytochrome b5 reductase gene leading to methemoglobinemia type I Blood, February 15, 2001; 97(4): 1106 - 1114. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, wild type; 
,
mutant. (A) Samples were incubated for 10 minutes at various
temperatures. (B) Incubation at 37°C for various times. (C)
Incubation for various times at 37°C with 1.5 U/µL trypsin.
cytochrome b5 reductase.
J Biochem.
1989;105:312