Effects of AGPAT4 Gene Interference and Overexpression on Lipid Metabolism in Mammary Epithelial Cells of Xinong Saanen Dairy Goats (Capra hircus)
YAO Wei-Wei, LUO Jun*, WANG Xin-Pei, QU Xiao-Peng, TIAN Hui-Bin, NIU Hui-Min
College of Animal Science and Technology, Northwest A&F University/Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture, Yangling 712100, China
Abstract：AGPAT4 (1-acylglycerol-3-phosphate O-acyltransferase 4) is a member of the 1-acylglycerol-3-phosphate O acyltransferase family of enzymes that catalyze synthesis of the triacylglycerol (TAG). In order to clarify the gene sequence and protein structure of AGPAT4 and to preliminarily analyze the effect of AGPAT4 on lipid metabolism in goat mammary epithelial cells (GMECs), In this study, the mammary epithelial cells of Xinong Saanen dairy goat (Capra hircus) were used as experimental materials, the AGPAT4 gene was cloned by reverse transcription-PCR (RT-PCR). Based on the cloned sequence, the bioinformatics analysis was conducted using online website and tools. qPCR was applied to detect the expression of GMECs lipid metabolism-related genes after AGPAT4 interference and overexpression. Then, the content of triacylglycerol in GMECs was detected after interference or overexpression of AGPAT4. The results showed that the CDS region of AGPAT4 gene was successfully cloned, with 1 137 bp (sequence ID: XM_005684959.3), encoding 378 amino acids, protein molecular weight of 43 939.35 u, theoretical pI value of 8.92, transmembrane structure and no signal peptide, which was an unstable protein with positive charge. The AGPAT4 protein interacted with AGPAT1, AGPAT2, lipin3 (LPIN3), diacylglycerol kinases (DGK), phospholipase D (PLD), cytidine diphosphate-diacylglycerol synthase (CDS) and other proteins. The downregulation of AGPAT4 via mRNA interference caused a significant reduction in acetyl coa carboxylase (ACC) gene expression (P<0.05). Overexpression of AGPAT4 significantly up-regulated the expression of stearoyl-coenzyme A desaturase (SCD1) and ACC genes (P<0.05) and significantly promoted the accumulation of intracellular TAG (P<0.05). These results indicated that AGPAT4 gene had positive regulation on lipid synthesis in GMECs. This study provides basic data for further research on the regulatory mechanism of AGPAT4 gene on milk fat metabolism of goat milk.
[1] 翁晨, 邓泽元, 李静. 2020. 乳制品脂质组成的比较[J]. 食品科学, 41(04): 149-156. (Weng C, Deng Z Y, Li J.2020. Comparison of lipid composition of dairy products[J]. Food Science, 41(04): 149-156.) [2] Agarwal A K, Sukumaran S, Cortés V A, et al.2011. Human 1-acylglycerol-3-phosphate O-acyltransferase isoforms 1 and 2: Biochemical characterization and inability to rescue hepatic steatosis in Agpat2(-/-) gene lipodystrophic mice[J]. The Journal of Biological Chemistry, 286(43): 37676-37691. [3] Agarwal A K, Tunison K, Dalal J S, et al.2017. Metabolic, reproductive, and neurologic abnormalities in agpat1-null mice[J]. Endocrinology, 158(11): 3954-3973. [4] Andreas N J.2015. Kampmann and Mehring. Human breast milk: A review on its composition and bioactivity[J]. Early Human Development, 91(11): 629-635. [5] Barber M C, Price N T, Travers M T.2005. Structure and regulation of acetyl-CoA carboxylase genes of metazoa[J]. Biochimica et Biophysica Acta, 1733(1): 1-28. [6] Bradley R M, Bloemberg D, Aristizabal J J, et al.2018. Lpaatδ/Agpat4 deficiency impairs maximal force contractility in soleus and alters fibre type in extensor digitorum longus muscle[J]. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 1863(7): 700-711. [7] Bradley R M, Duncan R E.2018. The lysophosphatidic acid acyltransferases (acylglycerophosphate acyltransferases) family: One reaction, five enzymes, many roles[J]. Current Opinion in Lipidology, 29(2): 110-115. [8] Bradley R M, Marvyn P M, Aristizabal Henao J J, et al.2015. Acylglycerophosphate acyltransferase 4 (AGPAT4) is a mitochondrial lysophosphatidic acid acyltransferase that regulates brain phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol levels[J]. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 1851(12): 1566-1576. [9] Bradley R M, Mardian E B, Bloemberg D, et al.2017. Mice deficient in lysophosphatidic acid acyltransferase delta (Lpaatδ)/acylglycerophosphate acyltransferase 4 (Agpat4) have impaired learning and memory[J]. Molecular and Cellular Biology, 37(22): e217-e245. [10] Broadfield L A, Pane A A, TalebiA, et al.2021. Lipid metabolism in cancer: New perspectives and emerging mechanisms[J]. Developmental Cell, 56(10): 1363-1393. [11] Eto M, Shindou H, Shimizu T.2014. A novel lysophosphatidic acid acyltransferase enzyme (LPAAT4) with a possible role for incorporating docosahexaenoic acid into brain glycerophospholipids[J]. Biochemical and Biophysical Research Communications, 443(2): 718-724. [12] Hishikawa D, Yanagida K, Nagata K, et al.2020. Hepatic levels of DHA-containing phospholipids instruct SREBP1-mediated synthesis and systemic delivery of polyunsaturated fatty acids[J]. iScience, 23(9): 101495. [13] Isaacs C E, Litov R E, Thormar H.1995. Antimicrobial activity of lipids added to human milk, infant formula, and bovine milk[J]. Journal Of Nutritional Biochemistry, 6(7): 362-366. [14] Karagiota A, Chachami G, Paraskeva E.2022. Lipid metabolism in cancer: The role of acylglycerolphosphate acyltransferases (AGPATs)[J]. Cancers, 14(1): 228. [15] Korbes A P, Kulcheski F R, Margis R, et al.2016. Molecular evolution of the lysophosphatidic acid acyltransferase (LPAAT) gene family[J]. Molecular Phylogenetics and Evolution, 96: 55-69. [16] Lopez C, Menard O.2011. Human milk fat globules: Polar lipid composition and in situ structural investigations revealing the heterogeneous distribution of proteins and the lateral segregation of sphingomyelin in the biological membrane[J]. Colloids and Surfaces B: Biointerfaces, 83(1): 29-41. [17] Lu B, Jiang Y J, Zhou Y L, et al.2005. Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart[J]. The Biochemical Journal, 385(Pt 2): 469-477. [18] Mardian E B, Bradley R M, Aristizabal J J, et al.2017. Agpat4/Lpaatdelta deficiency highlights the molecular heterogeneity of epididymal and perirenal white adipose depots[J]. Journal Of Lipid Research, 58(10): 2037-2050. [19] Pagliuso A, Valente C, Giordano L L, et al.2016. Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase delta[J]. Nature Communications, 7: 12148. [20] Prasad S S, Garg A, Agarwal A K.2011. Enzymatic activities of the human AGPAT isoform 3 and isoform 5: Localization of AGPAT5 to mitochondria[J]. Journal of Lipid Research, 52(3): 451-462. [21] Ruggles K V, Turkish A, Sturley S L.2013. Making, baking, and breaking: The synthesis, storage, and hydrolysis of neutral lipids[J]. Annual Review of Nutrition, 33(1): 413-451. [22] Schmidt J A, Brown W J.2009. Lysophosphatidic acid acyltransferase 3 regulates Golgi complex structure and function[J]. The Journal of Cell Biology, 186(2): 211-218. [23] Takeuchi K, Reue K.2009. Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis[J]. American Journal of Physiology Endocrinology and Metabolism, 296(6): E1195-E1209. [24] Wang H, Airola M V, Reue K.2017. How lipid droplets "TAG" along: Glycerolipid synthetic enzymes and lipid storage[J]. Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids, 1862(10 Pt B): 1131-1145. [25] Welte M A.2009. Fat on the move: Intracellular motion of lipid droplets[J]. Biochemical Society Transactions, 37(Pt 5): 991-996. [26] Zhukovsky M A, Filograna A, Luini A, et al.2019. The structure and function of acylglycerophosphate acyltransferase 4/lysophosphatidic acid acyltransferase delta (AGPAT4/LPAATδ)[J]. Frontiers in Cell and Developmental Biology, 7: 147.
