MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF HUMAN ACYL-COENZYME-A CHOLESTEROL ACYLTRANSFERASE CDNA IN MUTANT CHINESE-HAMSTER OVARY CELLS

Accumulation of cholesterol esters as cytoplasmic lipid droplets withi n macrophages and smooth muscle cells is a characteristic feature of e arly lesions of atherosclerotic plaque. Intracellularly, an essential element in forming cholesterol ester from cholesterol is the enzyme ac yl-coenzyme A:cholesterol acyltransferase (ACAT). ACAT is a membrane p rotein located in the endoplasmic reticulum. The ACAT protein has neve r been purified to homogeneity, and no antibodies directed against ACA T have been reported. The gene(s) encoding this enzyme had not been is olated. This laboratory had previously reported the isolation of Chine se hamster ovary cells expressing human ACAT activity. From DNAs of th ese cells, we have cloned a 1.2-kb exonic human genomic DNA. This led to the eventual cloning of a 4-kb cDNA clone (K1) from a human macroph age cDNA library. Transfection of K1 in ACAT-deficient mutant Chinese hamster ovary cells complemented the mutant defect and resulted in the expression of human ACAT activity. K1 contained an open reading frame of 1650 bp encoding an integral membrane protein of 550 amino acids. Protein homology analysis showed that the predicted K1 protein shared homologous peptide sequences with other enzymes involved in the cataly sis of acyl adenylate formation followed by acyl thioester formation a nd acyl transfer. These results indicate that K1 encodes a structural gene for ACAT. The cDNA reported here should facilitate future molecul ar studies on ACAT.