title: Purification, cloning, and expression of a human enzyme with acyl coenzyme A: cholesterol acyltransferase activity, which is identical to liver carboxylesterase.
abstract: An enzyme with acyl coenzyme A:cholesterol acyltransferase (ACAT) activity was isolated from porcine liver, and sequences derived from trypsinized peptides indicated homology to liver carboxylesterase. By use of degenerate primers, human cDNA clones were identified, which were identical to human liver carboxylesterase. Expression of the full-length cDNA in Chinese hamster ovary (CHO) cells led to an approximately threefold increase in cellular ACAT activity. This was accompanied by an approximately 20-fold increase of cellular cholesteryl ester content. By light and electron microscopy, recombinant CHO cells contained numerous lipid droplets that were not present in control CHO cells. Expression of an antisense cDNA in HepG2 cells reduced cellular ACAT activity by 35% compared with control. To further investigate the role of the enzyme in cellular cholesterol homeostasis, regulation of the mRNA was investigated in 7-day cultured human mononuclear phagocytes (MNPs). When these cells were incubated in lipoprotein-deficient serum for 18 hours, the mRNA for ACAT/carboxylesterase was almost not detectable on Northern blots, whereas after incubation with acetylated low-density lipoproteins, a strong hybridization signal was obtained. This is evidence that the mRNA of ACAT/carboxylesterase is induced by cholesterol loading. It is concluded from the data presented that ACAT/carboxylesterase is relevant for cellular cholesterol esterification in vivo. The regulation in MNPs indicates that the enzyme is also involved in foam cell formation during early atherogenesis.
authors.affiliation: Institute for Clinical Chemistry, University of Regensburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Becker
authors.forename: A
authors.initials: A
authors.lastname: Böttcher
authors.forename: K J
authors.initials: KJ
authors.lastname: Lackner
authors.forename: P
authors.initials: P
authors.lastname: Fehringer
authors.forename: F
authors.initials: F
authors.lastname: Notka
authors.forename: C
authors.initials: C
authors.lastname: Aslanidis
authors.forename: G
authors.initials: G
authors.lastname: Schmitz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000201:enzymology; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D016373:DNA, Antisense / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA; D002785:Sterol O-Acyltransferase / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D014407:Tumor Cells, Cultured / Q000737:chemistry
chemical_list: D016373:DNA, Antisense; D012333:RNA, Messenger; D002785:Sterol O-Acyltransferase; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase