Abstract
Background: 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. Methods: The 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). Results: Lentivirus-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. Conclusions: Results suggest that BTN1A1 plays an important role in regulating LD synthesis via a mechanism involving membrane phospholipid composition.
Original language | English (US) |
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Article number | 72 |
Journal | Journal of Animal Science and Biotechnology |
Volume | 11 |
Issue number | 1 |
DOIs | |
State | Published - Jul 3 2020 |
Keywords
- Lipid droplet
- Mammary epithelial cell
- Milk fat globule
- Phospholipid
ASJC Scopus subject areas
- Biotechnology
- Food Science
- Biochemistry
- Animal Science and Zoology