Abstract
The present study demonstrates that two different forms of the intracellular cholesterol esterification enzyme acyl-CoA:cholesterol acyltransferase (ACAT) are present in the nonhuman primate hepatocyte; one is similar to that originally cloned from human genomic DNA, here termed ACAT1, while a second gene product, termed ACAT2, is reported here. The primate ACAT2 gene product was cloned from an African green monkey liver cDNA library. Sequence analysis of an isolated, full-length clone of ACAT2 cDNA identified an open reading frame encoding a 526-amino acid protein with essentially no sequence similarity to the ACAT1 cDNA over the N-terminal 101 amino acids but with 57% identity predicted over the remaining 425 amino acids. Transfection of the cloned ACAT2 cDNA into two different mammalian cell types resulted in the production of abundant ACAT activity which was sensitive to ACAT inhibitors. Northern blot analysis showed that the ACAT2 mRNA was expressed primarily in liver and intestine in monkeys. In contrast, ACAT1 mRNA was expressed in almost all tissues examined. Topologic predictions from the amino acid sequence of ACAT2 indicates that it has seven trans-membrane domains in a configuration that places the putative active site of the enzyme in the lumen of the endoplasmic reticulum. This orientation of ACAT2 in the endoplasmic reticulum membrane, in addition to its expression only in liver and intestine, suggests that this enzyme may have as a primary function, the secretion of cholesteryl esters into apoB-containing lipoproteins.
Highlights
The intracellular formation of cholesteryl esters catalyzed by the action of the enzyme acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26)1 appears to be nearly ubiqitous in mammalian cells [1]
Cloning and Sequence Analysis—As the initial step in the cDNA cloning strategy, reverse transcriptase-polymerase chain reaction (RT-PCR) using monkey liver mRNA as template was performed with primer sequences whose design was based on a partial human ACAT-related gene sequence termed ARGP2 by Sturley et al [17]
Evidence in nonhuman primate models has been obtained over the past two decades indicating that the accumulation of cholesteryl esters in low density lipoprotein, with the associated low density lipoprotein particle enlargement, is highly correlated with the extent of coronary artery atherosclerosis [35,36,37,38]
Summary
Materials—For earlier experimental protocols [19], organs from adult male African green monkeys (Cercopithecus aethiops) were collected at the time of necropsy, cut into 1-cm cubes, and snap frozen in liquid nitrogen. Library Screening—An African green monkey liver, oligo(dT)-primed cDNA library was constructed in the pcDNAII plasmid vector by Invitrogen (San Diego) using RNA purified in our laboratory. Avian myeloblastosis virus reverse transcriptase (Promega) with an oligo(dT) primer was used to synthesize first strand cDNA from 0.5 g of poly(A) mRNA which had been isolated from African green monkey liver biopsy tissue as described below. ACAT1 cDNA Isolation—A cDNA homolog of the original human ACAT1 clone [2] was isolated from African green monkey and cynomolgus monkey adrenal poly(A) mRNA using an oligo(dT)-primed RT-PCR with primers designed from the published human ACAT sequence. The analytical methods used for the probability projections of transmembrane helices [32] are based on multiple sequence alignments with proteins whose crystal structures are recorded in the SWISS-PROT data base
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