Abstract
BackgroundMilk lipids originate from cytoplasmic lipid droplets (LD) that are synthesized and secreted from mammary epithelial cells by a unique membrane-envelopment process. Butyrophilin 1A1 (BTN1A1) is one of the membrane proteins that surrounds LD, but its role in bovine mammary lipid droplet synthesis and secretion is not well known.MethodsThe objective was to knockout BTN1A1 in bovine mammary epithelial cells (BMEC) via the CRISPR/Cas9 system and evaluate LD formation, abundance of lipogenic enzymes, and content of cell membrane phospholipid (PL) species. Average LD diameter was determined via Oil Red O staining, and profiling of cell membrane phospholipid species via liquid chromatography-tandem mass spectrometry (LC-MS/MS).ResultsLentivirus-mediated infection of the Cas9/sgRNA expression vector into BMEC resulted in production of a homozygous clone BTN1A1(−/−). The LD size and content decreased following BTN1A1 gene knockout. The mRNA abundance of fatty acid synthase (FASN) and peroxisome proliferator-activated receptor-gamma (PPARG) was downregulated in the BTN1A1(−/−) clone. Subcellular analyses indicated that BTN1A1 and LD were co-localized in the cytoplasm. BTN1A1 gene knockout increased the percentage of phosphatidylethanolamine (PE) and decreased phosphatidylcholine (PC), which resulted in a lower PC/PE ratio.ConclusionsResults suggest that BTN1A1 plays an important role in regulating LD synthesis via a mechanism involving membrane phospholipid composition.
Highlights
The process of milk fat secretion begins in the endoplasmic reticulum of mammary epithelial cells (MEC) from which lipid droplets (LD) begin to form [1]
It has been suggested that intracellular LD formed by mammary epithelial cells are related to the size of secreted milk fat globules (MFG) [9, 10] and, the mechanism regulating LD size in the cytoplasm is relevant to the properties of MFG
Butyrophilin 1A1 (BTN1A1) protein and lipid droplet co-localization The BTN1A1-GFP fusion protein led to green fluorescence in the bovine mammary epithelial cells (BMEC) (Fig. 1a)
Summary
The process of milk fat secretion begins in the endoplasmic reticulum of mammary epithelial cells (MEC) from which lipid droplets (LD) begin to form [1]. The BTN1A1 protein accounts for more than 20% of total protein in the MFGM [3, 16] Abundance of these proteins increases during lactation in the bovine mammary gland [17]. Milk lipids originate from cytoplasmic lipid droplets (LD) that are synthesized and secreted from mammary epithelial cells by a unique membrane-envelopment process. Butyrophilin 1A1 (BTN1A1) is one of the membrane proteins that surrounds LD, but its role in bovine mammary lipid droplet synthesis and secretion is not well known
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