Two Human ACAT2 mRNA Variants Produced by Alternative Splicing and Coding for Novel Isoenzymes

Abstract

Acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2) plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms, named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 (ACAT2b mRNA) and exons 4-5 plus 8-9-10 (ACAT2c mRNA). Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RT-PCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestinecells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.

Analysis of acyl coenzyme A: cholesterol acyltransferase (ACAT) expression in human tissues

“…However no ACAT-2 mRNA was detected in adult liver (Yao et al,2005), in agreement with previous reports of low levels of ACAT-2 expression in adult human liver compared to foetal liver (cf. also Chang et al, 2000).…”

“…Whereas the full length ACAT-2a mRNA encodes a deduced ~60 kDa protein consisting of 522 amino acids, the ACAT-2b and ACAT-2c variants encode proteins of 502 amino acids and 379 amino acids, respectively. ACAT-2b mRNA differs from ACAT-2a mRNA by lacking exon 4, while ACAT-2c mRNA differs from ACAT-2a mRNA by lacking exons 4, 5, and 8, 9 and 10 (Yao et al,2005).…”

“…Chang et al (2000) reported that human foetal liver (as well as cultured HepG2 and Caco-2 cells) expressed ACAT-2 as a single ~46 kDa protein. Yao et al (2005) Oelkers et al (1998) noting that ACAT-2 has two putative N-linked glycosylation sites, we are not aware of any reports of actual ACAT-2 glycosylation. It is noted that the commercial supplier of an anti-ACAT-2 antibody reported that their preparation could detect a 60 kDa protein in human fibroblasts, a 49 kDa protein in human Jurkat cell lysates, and 60 kDa proteins in mouse and rat liver, intestines and fibroblasts (https://www.caymanchem.com/app/template/Product.vm/catalog/100027).…”

“…ACAT-2 expression products are particularly abundant in the villi apices of the small intestine (enterocytes) and also in fetal hepatocytes and various cultured human cell lines such as HepG2 and Caco-2 (Yao et al, 2005). However ACAT-2 expression levels diminished in hepatocytes as children aged and was virtually undetectable in adults .…”

Low-level expression of human <italic>ACAT2</italic> gene in monocytic cells is regulated by the C/EBP transcription factors

“…The regulatory expression and functional mechanisms of human ACAT1 have been studied [15][16][17][18][19][20][21][22]. For human ACAT2 gene, we have previously reported its genomic organization, the intestinal Caco-2 cell differentiation-dependent promoter activity, and two novel isoforms (named as ACAT2b and ACAT2c) encoded by the alternative-spliced two mRNA variants with reduced enzymatic activities [23,24]. Moreover, it has been reported that ACAT2 is highly expressed in the livers of mice and monkeys [25][26][27].…”