Anti-AGPAT6 monoclonal antibody Made to order

Most cells synthesize their glycerophospholipids and triglycerides (TG) to maintain the cellular integrity and to provide energy for cellular functions. The phospholipids are synthesized de novo in cells through an evolutionary conserved process involving serial acylations of glycerol-3-phosphate. Several isoforms of the enzyme 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT) acylate lysophosphatidic acid at the sn-2 position to produce phosphatidic acid. We cloned a cDNA predicted to be an AGPAT isoform and designated it AGPAT9. The human AGPAT9 gene spans across 14 exons and encodes for a polypeptide of 534 amino acids. AGPAT9 is highly expressed in the lung and spleen, followed by leukocyte, omental adipose tissue, and placenta. In the Chinese Hamster Ovary (CHO), cell lysates overexpressing AGPAT9, we observed AGPAT activity but not the lysophosphatidylcholine acyltransferase activity. When AGPAT9 is coexpressed with AGPAT1 in CHO cells, both the isoforms localize to the endoplasmic reticulum (ER) and occupy the same ER domain as AGPAT1. Despite substitution of asparagine with proline in the NHX4D motif and arginine with cysteine in the EGTR motif, AGPAT9 retains AGPAT activity suggesting that residues asparagine and arginine in the NHX4D and EGTR motifs respectively are not essential for the enzymatic activity. Based on the X-ray crystallographic structure of a related acyltransferase, squash gpat, a model is proposed in which a hydrophobic pocket in AGPAT9 accommodates fatty acyl chains of both substrates in an orientation, whereas the HX4D motif participates in catalysis. Based on the activity and expression pattern of AGPAT9 in the lung and spleen, this novel isoform could be implicated in the biosynthesis of phospholipids and TG in these tissues.

Sterol regulatory element binding protein 1 (SREBP-1) is well-known as the master regulator of lipogenesis in rodents. Acyl-CoA synthetase short-chain family member 2 (ACSS2) plays a key role in lipogenesis by synthesizing acetyl-CoA from acetate for lipogenesis. ATP citrate lyase (ACLY) catalyzes the conversion of citrate and coenzyme A to acetyl-CoA, hence, it is also important for lipogenesis. Although ACSS2 function in cancer cells has been elucidated, its essentiality in ruminant mammary lipogenesis is unknown. Furthermore, ACSS2 gene promoter and its regulatory mechanisms have not known. Expression of ACSS2 was high in lipid synthesizing tissues, and its expression increased during lactation compared with non-lactating period. Simultaneous knockdown of both ACSS2 and ACLY by siRNA in primary goat mammary epithelial cells decreased (p<0.05) the mRNA abundance of genes associated with de novo fatty acid synthesis (FASN, ACACA, SCD1) and triacylglycerol (TAG) synthesis (DGAT1, DGAT2, GPAM, and AGPAT6). Genes responsible for lipid droplet formation and secretion (PLIN2 and PLIN3) and fatty acid oxidation (ATGL, HSL, ACOX, and CPT1A) all decreased (p<0.05) after ACSS2 and ACLY knockdown. Total cellular TAG content and lipid droplet formation also decreased. Use of a luciferase reporter assay revealed a direct regulation of ACSS2 by SREBP-1. Furthermore, SREBP-1 interacted with an SRE (SREBP response element) spanning at -475 to -483bp on the ACSS2 promoter. Taken together, our results revealed a novel pathway that SREBP-1 may regulate fatty acid and TAG synthesis by regulating the expression of ACSS2.