AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase

Abstract

AGPAT6 is a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family that appears to be important in triglyceride biosynthesis in several tissues, but the precise biochemical function of the enzyme is unknown. In the current study, we show that AGPAT6 is a microsomal glycerol-3-phosphate acyltransferase (GPAT). Membranes from HEK293 cells overexpressing human AGPAT6 had higher levels of GPAT activity. Substrate specificity studies suggested that AGPAT6 was active against both saturated and unsaturated long-chain fatty acyl-CoAs. Both glycerol 3-phosphate and fatty acyl-CoA increased the GPAT activity, and the activity was sensitive to N-ethylmaleimide, a sulfhydryl-modifying reagent. Purified AGPAT6 protein possessed GPAT activity but not AGPAT activity. Using [ 13 C 7 ]oleic acid labeling and mass spectrometry, we found that overexpression of AGPAT6 increased both lysophosphatidic acid and phosphatidic acid levels in cells. In these studies, total triglyceride and phosphatidylcholine levels were not significantly altered, although there were significant changes in the abundance of specific phosphatidylcholine species. Human AGPAT6 is localized to endoplasmic reticulum and is broadly distributed in tissues. Membranes of mammary epithelial cells from Agpat6-deficient mice exhibited markedly reduced GPAT activity compared with membranes from wildtype mice. Reducing AGPAT6 expression in HEK293 cells through small interfering RNA knockdown suggested that AGPAT6 significantly contributed to HEK293 cellular GPAT activity. Our data indicate that AGPAT6 is a microsomal GPAT, and we propose renaming this enzyme GPAT4.

Molecular cloning, sequence characterization, and gene expression profiling of a novel water buffalo (Bubalus bubalis) gene, AGPAT6

“…It contains 1371 nucleotides encoding a putative protein of 456 aa, 52 kDa in size, with a pI of 9.34. As in previous reports of human and mouse, the AGPAT6 protein in water buffalo also contains three hydrophobic transmembrane regions and a signal peptide of 37 aa, which implies that it is a transmembrane protein Chen et al, 2008;Nagle et al, 2008). Generally, most protein functions are regulated by phosphorylation/dephosphorylation.…”

“…AGPAT6 is broadly expressed and can be detected at the mRNA level in multiple tissues examined Agarwal et al, 2007;Chen et al, 2008). In mouse, AGPAT6 is expressed at a high level in brown adipose tissue, white adipose tissue, liver, and mammary epithelium of breast tissue Bionaz and Loor, 2008a).…”

“…So far, eight members of the AGPAT gene family have been described in humans, which are AGPAT1-8, and each of them possesses a lysophosphatidic acid acyltransferase motif (Ye et al, 2005;Nagle et al, 2008). As a member of the AGPAT family, AGPAT6 appears to play a key role in lipid biosynthesis (Chen et al, 2008). After careful examination of the AGPAT enzyme activity by Chen et al (2008) and Nagle et al (2008), the enzyme was recognized as another endoplasmic reticulum-localized glycerol phosphate acyltransferase (GPAT) and renamed as GPAT4.…”

“…After careful examination of the AGPAT enzyme activity by Chen et al (2008) and Nagle et al (2008), the enzyme was recognized as another endoplasmic reticulum-localized glycerol phosphate acyltransferase (GPAT) and renamed as GPAT4. The AGPAT6 gene has recently been identified in humans, mice, cattle, and chickens (Li et al, 2003;Chen et al, 2008;Zimin et al, 2009). In both human and mouse, the AGPAT6 gene consists of 13 exons, whereas that in cattle and chicken contains 12 exons.…”

“…In both human and mouse, the AGPAT6 gene consists of 13 exons, whereas that in cattle and chicken contains 12 exons. The AGPAT6 protein contains 456 amino acids (aa) in the species mentioned above, except chicken, in which it is composed of 455 aa (Li et al, 2003;Beigneux et al, 2006;Chen et al, 2008;Nagle et al, 2008).…”