Recombinant Acyl-CoA:cholesterol Acyltransferase-1 (ACAT-1) Purified to Essential Homogeneity Utilizes Cholesterol in Mixed Micelles or in Vesicles in a Highly Cooperative Manner

Abstract

Acyl-coenzyme A:cholesterol acyltransferase (ACAT)is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A. The first gene encoding the enzyme, designated as ACAT-1, was identified in 1993 through an expression cloning approach. We isolated a Chinese hamster ovary cell line that stably expresses the recombinant human ACAT-1 protein bearing an N-terminal hexahistidine tag. We purified this enzyme approximately 7000-fold from crude cell extracts by first solubilizing the cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, then proceeding with an ACAT-1 monoclonal antibody affinity column and an immobilized metal affinity column. The final preparation is enzymologically active and migrates as a single band at 54 kDa on SDS-polyacrylamide gel electrophoresis. Pure ACAT-1 dispersed in mixed micelles containing sodium taurocholate, phosphatidylcholine, and cholesterol remains catalytically active. The cholesterol substrate saturation curves of the enzyme assayed either in mixed micelles or in reconstituted vesicles are both highly sigmoidal. The oleoyl-coenzyme A substrate saturation curves of the enzyme assayed under the same conditions are both hyperbolic. These results support the hypothesis that ACAT is an allosteric enzyme regulated by cholesterol. Specific polyclonal anti-ACAT-1 IgGs have been produced (19 -21). Immunoblot and immunodepletion analyses show that the ACAT-1 protein is present in homogenates from various human cells and tissues as a single 50-kDa protein band in SDS-PAGE (19) Furthermore, immunodepletion experiments using anti-ACAT-1 IgGs suggest that the ACAT-1 protein plays a major role in ACAT catalysis in human fibroblasts, HepG2 cells, human hepatocytes, macrophages, adrenal glands, and kidneys (22). In contrast, in human intestines, approximately 80% of total measurable ACAT activity is resistant to immunodepletion, suggesting that ACAT activity in this particular tissue may be largely due to the presence of a different ACAT protein (22). Judging from results currently available, it is possible that the physiological functions of ACAT-1 and ACAT-2 are different in different species. Whether ACAT-2 is responsible for most of the observed ACAT activity in human intestines is not clear at present. Acyl-coenzymeThe ACAT protein has never before been purified to homogeneity. The difficulty in doing so was largely due to its minute quantity and the lack of a suitable detergent for solubilizing the protein from the endoplasmic reticulum membrane with retention of enzyme activity (reviewed in Ref. 5). As described in this article, we have isolated a CHO cell line (HisACAT-1 cells) that stably expresses the human ACAT-1 (hACAT-1) protein bearing a hexahistidine tag at its N terminus. We report the use of this cell line as the starting material to develop a purification scheme that enables us to purify the enzyme to essential homogenei...

Localization of Human Acyl-Coenzyme A:Cholesterol Acyltransferase-1 (ACAT-1) in Macrophages and in Various Tissues

“…This result is con- sistent with previous studies in various cells and tissues supporting the notion that the main mechanism for increase in ACAT enzyme activity by cholesterol influx is probably due to the allosteric property of the ACAT-1 enzyme in combination with an increase in intracellular cholesterol trafficking toward the endoplasmic reticulum. 1,28,29 The electron microscopic studies in macrophages described in this study report ACAT-1 localization at the ultrastructural level for the first time, demonstrating that the ACAT-1 protein is located mainly in the rough endoplasmic reticulum. This result is consistent with previous biochemical studies using other cell types, suggesting that ACAT-1 is an integral membrane protein that functions in the rough endoplasmic reticulum.…”

Niemann–Pick Type C Mutations Cause Lipid Traffic Jam

“…This limits the further acquisition of cholesterol by suppressing transcription of sterol-synthesizing genes, such as 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. Cholesterol arriving in the ER membrane also activates and serves as substrate for an ER enzyme, acyl-CoA/cholesterol acyltransferase, which catalyzes the esterification of a fatty acid to cholesterol for storage in cytoplasmic lipid droplets (20,21). Thus, cellular-free cholesterol levels are kept within a tight range.…”

Human Acyl-CoA:Cholesterol Acyltransferase-1 in the Endoplasmic Reticulum Contains Seven Transmembrane Domains

“…Expression of this cDNA in insect cells devoid of endogenous ACAT activity produced ample ACAT activity in vitro (15). The recombinant human ACAT-1 (hA-CAT-1) protein expressed in CHO cells has recently been purified to homogeneity with retention in catalytic activity (16). Human ACAT-1 gene localization and organization have been reported (17)(18).…”

“…Through genetic engineering, this region was fused in frame with bacterial glutathione S-transferase to produce a glutathione S-transferase-hACAT-1 fusion protein; the purified fusion protein was used as antigen to produce high titer, specific anti-hACAT-1 rabbit polyclonal antibodies (designated as DM-10) (19). Western blotting with DM 10 (15,16,19) could detect the hACAT-1 protein in various human cells and tissues as a single 50-kDa protein. Immunodepletion experiments using this anti-ACAT-1 IgG suggest that the hACAT-1 protein plays a major role in ACAT catalysis in human fibroblasts, HepG2 cells, hepatocytes, macrophages, adrenal glands, and kidneys (19 -21).…”