Cloning and expression of a human ATP-citrate lyase cDNA.
A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.
Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
N A
NA
Elshourbagy
J C
JC
Near
P J
PJ
Kmetz
T N
TN
Wells
P H
PH
Groot
B A
BA
Saxty
S A
SA
Hughes
M
M
Franklin
I S
IS
Gloger
D001275:ATP Citrate (pro-S)-Lyase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D000882:Haplorhini; D006801:Humans; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D001275:ATP Citrate (pro-S)-Lyase