PURIFICATION, CLONING, AND EXPRESSION OF A HUMAN ENZYME WITH ACYL-COENZYME-A - CHOLESTEROL ACYLTRANSFERASE ACTIVITY, WHICH IS IDENTICAL TO LIVER CARBOXYLESTERASE

An enzyme with acyl coenzyme A:cholesterol acyltransferase (ACAT) acti vity was isolated from porcine liver, and sequences derived from tryps inized peptides indicated homology to liver carboxylesterase. By use o f degenerate primers, human cDNA clones were identified, which were id entical to human liver carboxylesterase. Expression of the full-length cDNA in Chinese hamster ovary (CHO) cells led to an approximately thr eefold increase in cellular ACAT activity. This was accompanied by an approximate to 20-fold increase of cellular cholesteryl ester content. By light and electron microscopy, recombinant CHO cells contained num erous lipid droplets that were not present in control CHO cells. Expre ssion of an antisense cDNA in HepG2 cells reduced cellular ACAT activi ty by 35% compared with control. To further investigate the role of th e enzyme in cellular cholesterol homeostasis, regulation of the mRNA w as 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 o n Northern blots, whereas after incubation with acetylated low-density lipoproteins, a strong hybridization signal was obtained. This is evi dence that the mRNA of ACAT/carboxylesterase is induced by cholesterol loading. It is concluded from the data presented that ACAT/carboxyles terase is relevant for cellular cholesterol esterification in vivo. Th e regulation in MNPs indicates that the enzyme is also involved in foa m cell formation during early atherogenesis.