Dataset Information

Clinical, metabolic, and genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in cats.

ABSTRACT: Two non-pedigreed male castrated cats had persistent cyanosis over a 3-year observation period. Clinical cardiopulmonary evaluations did not reveal abnormalities, but the blood remained dark after exposure to air. Erythrocytic methemoglobin concentrations were high (~40% of hemoglobin) and cytochrome b5 reductase (CYB5R) activities in erythrocytes were low (?15% of control). One cat remained intolerant of exertion, and the other cat developed anemia and died due to an unidentified comorbidity. Whole-genome sequencing revealed a homozygous c.625G>A missense variant (B4:137967506) and a c.232-1G>C splice acceptor variant (B4:137970815) in CYB5R3, respectively, which were absent in 193 unaffected additional cats. The p.Gly209Ser missense variant likely disrupts a nicotinamide adenine dinucleotide (NADH)-binding domain, while the splicing error occurs at the acceptor site for exon 4, which likely affects downstream translation of the protein. The 2 novel CYB5R3 variants were associated with methemoglobinemia using clinical, biochemical, genomics, and in silico protein studies. The variant prevalence is unknown in the cat population.

SUBMITTER: Jaffey JA

PROVIDER: S-EPMC6872605 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Clinical, metabolic, and genetic characterization of hereditary methemoglobinemia caused by cytochrome b<sub>5</sub> reductase deficiency in cats.

Jaffey Jared A JA Reading N Scott NS Giger Urs U Abdulmalik Osheiza O Buckley Ruben M RM Johnstone Sophie S Lyons Leslie A LA

Journal of veterinary internal medicine 20191025 6

Two non-pedigreed male castrated cats had persistent cyanosis over a 3-year observation period. Clinical cardiopulmonary evaluations did not reveal abnormalities, but the blood remained dark after exposure to air. Erythrocytic methemoglobin concentrations were high (~40% of hemoglobin) and cytochrome b<sub>5</sub> reductase (CYB5R) activities in erythrocytes were low (≤15% of control). One cat remained intolerant of exertion, and the other cat developed anemia and died due to an unidentified com ...[more]

PMID: 31650629

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Project description:Membrane cytochrome b5 reductase is a pleiotropic oxidoreductase that uses primarily soluble reduced nicotinamide adenine dinucleotide (NADH) as an electron donor to reduce multiple biological acceptors localized in cellular membranes. Some of the biological acceptors of the reductase and coupled redox proteins might eventually transfer electrons to oxygen to form reactive oxygen species. Additionally, an inefficient electron transfer to redox acceptors can lead to electron uncoupling and superoxide anion formation by the reductase. Many efforts have been made to characterize the involved catalytic domains in the electron transfer from the reduced flavoprotein to its electron acceptors, such as cytochrome b5, through a detailed description of the flavin and NADH-binding sites. This information might help to understand better the processes and modifications involved in reactive oxygen formation by the cytochrome b5 reductase. Nevertheless, more than half a century since this enzyme was first purified, the one-electron transfer process toward potential electron acceptors of the reductase is still only partially understood. New advances in computational analysis of protein structures allow predicting the intramolecular protein dynamics, identifying potential functional sites, or evaluating the effects of microenvironment changes in protein structure and dynamics. We applied this approach to characterize further the roles of amino acid domains within cytochrome b5 reductase structure, part of the catalytic domain, and several sensors and structural domains involved in the interactions with cytochrome b5 and other electron acceptors. The computational analysis results allowed us to rationalize some of the available spectroscopic data regarding ligand-induced conformational changes leading to an increase in the flavin adenine dinucleotide (FAD) solvent-exposed surface, which has been previously correlated with the formation of complexes with electron acceptors.

Project description:Heterocyclic and aromatic amine carcinogens are thought to lead to tumor initiation via the formation of DNA adducts, and bioactivation to arylhydroxylamine metabolites is necessary for reactivity with DNA. Carcinogenic arylhydroxylamine metabolites are cleared by a microsomal, NADH-dependent, oxygen-insensitive reduction pathway in humans, which may be a source of interindividual variability in response to aromatic amine carcinogens. The purpose of this study was to characterize the identity of this reduction pathway in human liver. On the basis of our findings with structurally similar arylhydroxylamine metabolites of therapeutic drugs, we hypothesized that the reductive detoxification of arylhydroxylamine carcinogens was catalyzed by NADH cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5). We found that reduction of the carcinogenic hydroxylamines of the aromatic amine 4-aminobiphenyl (4-ABP; found in cigarette smoke) and the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP; found in grilled meats) was indeed catalyzed by a purified system containing only human b5R and cyt b5. Specific activities were 56-346-fold higher in the purified system as compared to human liver microsomes (HLM), with similar Michaelis-Menten constants (K(m) values) in both systems. The stoichiometry for b5R and cyt b5 that yielded the highest activity in the purified system was also similar to that found in native HLM ( approximately 1:8 to 1:10). Polyclonal antisera to either b5R or cyt b5 significantly inhibited N-hydroxy-4-aminobiphenyl (NHOH-4-ABP) reduction by 95 and 89%, respectively, and immunoreactive cyt b5 protein content in individual HLM was significantly correlated with individual reduction of both NHOH-4-ABP and N-hydroxy-PhIP (NHOH-PhIP). Finally, titration of HLM into the purified b5R/cyt b5 system did not enhance the efficiency of reduction activity. We conclude that b5R and cyt b5 are together solely capable of the reduction of arylhydroxylamine carcinogens, and we further hypothesize that this pathway may be a source of individual variability with respect to cancer susceptibility following 4-ABP or PhIP exposure.

Dataset Information

Clinical, metabolic, and genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in cats.

Publications

Clinical, metabolic, and genetic characterization of hereditary methemoglobinemia caused by cytochrome b<sub>5</sub> reductase deficiency in cats.

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