cDNA cloning of mutant catalase in acatalasemic beagle dog: single nucleotide substitution leading to thermal-instability and enhanced proteolysis of mutant enzyme.
The mutant catalase purified previously from acatalasemic dog liver was heat-labile but possessed normal activity, suggesting a mutation within the coding region distal from the catalytic site. The nucleotide and deduced amino acid sequences of acatalasemic beagle dog catalase were determined by analysis of cDNA obtained by 5'- and 3'-RACE and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. Comparative analysis of cDNA sequences of normal and acatalasemic dog catalases indicated a single nucleotide difference where alanine(327) (G macro CT) was substituted with threonine (ACT). The mutant catalase, which was overexpressed in COS-1 cells, was heat-labile as previously observed with the purified enzyme from acatalasemic dog liver, indicating that this amino acid substitution can lead to structural instability. No catalase protein and activity were detected by immunoblotting and spectrophotomeric assay in acatalasemic dog reticulocytes although almost the same level of mRNA expression as that in the normal reticulocytes was observed. Pulse-labeling and immunoprecipitation examination indicated that the level of catalase synthesis in the acatalasemic dog reticulocytes was almost the same (approximately 80%) as that in the normal reticulocytes. On the other hand, the synthesized mutant catalase in reticulocytes was rapidly degraded (t(1/2): 1.8 h) compared with the normal catalase (t(1/2): 14.0 h) and this degradation was almost completely inhibited by lactacystin (LC). These results suggested that the proteolytic degradation mediated most likely by proteasome might be involved in disposing of the mutant catalase in acatalasemic erythroid cells.
Nakamura
K
K
Drug Metabolism and Pharmacokinetics Research Laboratories, Sankyo Co. Ltd., 2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140-8710, Japan. kouiti@shina.sankyo.co.jp
Watanabe
M
M
Takanaka
K
K
Sasaki
Y
Y
Ikeda
T
T
D020642:Acatalasia / Q000201:enzymology* / Q000235:genetics; D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002374:Catalase / Q000097:blood / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D018076:DNA, Complementary; D004285:Dogs; D004795:Enzyme Stability; D006358:Hot Temperature; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex; D012156:Reticulocytes / Q000201:enzymology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D009097:Multienzyme Complexes; C067713:lactacystin; D002374:Catalase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine