Malonyl/Acetyltransferase (MAT) Knockout Decreases Triacylglycerol and Medium-Chain Fatty Acid Contents in Goat Mammary Epithelial Cells

Abstract

Malonyl/acetyltransferase (MAT) is a crucial functional domain of fatty acid synthase (FASN), which plays a vital role in the de novo synthesis of fatty acids in vivo. Milk fatty acids are secreted by mammary epithelial cells. Mammary epithelial cells are the units of mammary gland development and function, and it is a common model for the study of mammary gland tissue development and lactation. This study aimed to investigate the effects of MAT deletion on the synthesis of triacylglycerol and medium-chain fatty acids. The MAT domain was knocked out by CRISPR/Cas9 in the goat mammary epithelial cells (GMECs), and in MAT knockout GMECs, the mRNA level of FASN was decreased by approximately 91.19% and the protein level decreased by 51.83%. The results showed that MAT deletion downregulated the contents of triacylglycerol and medium-chain fatty acids (p < 0.05) and increased the content of acetyl-Coenzyme A (acetyl-CoA) (p < 0.001). Explicit deletion of MAT resulted in significant drop of FASN, which resulted in downregulation of LPL, GPAM, DGAT2, PLIN2, XDH, ATGL, LXRα, and PPARγ genes in GMECs (p < 0.05). Meanwhile, mRNA expression levels of ACC, FASN, DGAT2, SREBP1, and LXRα decreased following treatment with acetyl-CoA (p < 0.05). Our data reveals that FASN plays critical roles in the synthesis of medium-chain fatty acids and triacylglycerol in GMECs.

Keywords: CRISPR/Cas9; FASN; MAT; acetyl-CoA.

Figure 1

sgRNA design and screening. (A) Schematic illustration of the sites and sequence of sgRNA. Red is the PAM sequence. (B) Outline of the experimental protocol. Lip2000—transfection reagents; Puro—puromycin; ATV—cell dissociation solution. (C) Target fragment amplification and cutting efficiency. DL2000—DNA marker; NO.1—cells knockout by sgRNA 1; and others in the same way. NO.1 = 168 bp + 419 bp, NO.2 = 361 bp + 226 bp, NO.3 = 296 bp + 291 bp, NO.4 = 297 bp + 290 bp, NO.5 = 291bp + 296 bp, NO.6 = 328 bp + 259 bp. (D) Genotypes of sgRNA knockout. The first line was a wild-type sequence, with red indicating a sgRNA sequence and green indicating a PAM sequence, and the proportion of detected genotypes was displayed on the right side.

Figure 2

The bioinformatics analysis of MAT knockout efficiency. (A) Secondary structure analysis. NC—negative control cells; NO.6-1—a genotype of NO.6; NO.6-2—another genotype of NO.6; long vertical line—α-helix; middle vertical line—β-fold; Short vertical bar—irregular curl. (B) Tertiary structure analysis. From N to C, it is red, orange, yellow, green, cyan, blue, and purple, respectively.

Figure 3

The trial analysis of MAT knockout efficiency. (A) MAT knockout efficiency at mRNA level. NC—negative control cells; NO.1—cells knockout by sgRNA 1; and others in the same way. (B) Protein changes of FASN in knockout cells. KO—MAT-knockout group (C); quantitative analysis of protein; MAT-KO—MAT-knockout group; ** p < 0.01, *** p < 0.001.

Figure 4

Off-target detection. (A) High-probability targeting site information of sgRNA 6 sequence in goat genome. “OT”—off-target site; the yellow highlight represents a coincident target sequence; the red highlight represents a coincident PAM sequence. (B) Target site amplification. “ON”—target site; “OT”—off-target site; “WT”—wild-type; “KO”—knockout. (C) Enzyme digestion detection of off-target site.

Figure 5

Effects of MAT-knockout on genes related to fatty acid metabolism. (A) Fatty acid synthesis and triacylglycerol hydrolysis. (B) Triacylglycerol synthesis (C) Lipid droplet formation, secretion and hydrolysis. (D) Regulatory factors. NC—negative control group; MAT-KO—knockout group; values are means ± SEM for three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Acetyl-CoA suppresses the fatty acid anabolism. (A) Effect of MAT knockout on intracellular acetyl-CoA. NC-A—the control group added with acetyl-CoA; KO-A—the knockout group added with acetyl-CoA. (B–D) Effect of acetyl-CoA treatment on genes related to fatty acid synthesis. Values are means ± SEM for three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.