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. 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). 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. Results suggest that BTN1A1 plays an important role in regulating LD synthesis via a mechanism involving membrane phospholipid composition.

中文翻译：

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敲除酪蛋白亚家族 1 成员 A1 (BTN1A1) 会改变牛乳腺上皮细胞中的脂滴形成和磷脂成分。

乳脂来源于细胞质脂滴 (LD)，其通过独特的膜包膜过程从乳腺上皮细胞合成和分泌。Butyrophilin 1A1 (BTN1A1) 是围绕 LD 的膜蛋白之一，但其在牛乳腺脂滴合成和分泌中的作用尚不清楚。目的是通过 CRISPR/Cas9 系统敲除牛乳腺上皮细胞 (BMEC) 中的 BTN1A1，并评估 LD 形成、脂肪生成酶的丰度和细胞膜磷脂 (PL) 种类的含量。平均 LD 直径通过油红 O 染色测定，并通过液相色谱-串联质谱 (LC-MS/MS) 分析细胞膜磷脂种类。慢病毒介导的 Cas9/sgRNA 表达载体感染 BMEC 导致产生纯合克隆 BTN1A1(-/-)。BTN1A1 基因敲除后，LD 大小和含量降低。脂肪酸合酶 (FASN) 和过氧化物酶体增殖物激活受体-γ (PPARG) 的 mRNA 丰度在 BTN1A1(-/-) 克隆中下调。亚细胞分析表明 BTN1A1 和 LD 共定位于细胞质中。BTN1A1基因敲除增加了磷脂酰乙醇胺（PE）的百分比并降低了磷脂酰胆碱（PC），从而导致PC/PE比率降低。结果表明，BTN1A1 通过涉及膜磷脂组成的机制在调节 LD 合成中起重要作用。亚细胞分析表明 BTN1A1 和 LD 共定位于细胞质中。BTN1A1基因敲除增加了磷脂酰乙醇胺（PE）的百分比并降低了磷脂酰胆碱（PC），从而导致PC/PE比率降低。结果表明，BTN1A1 通过涉及膜磷脂组成的机制在调节 LD 合成中起重要作用。亚细胞分析表明 BTN1A1 和 LD 共定位于细胞质中。BTN1A1基因敲除增加了磷脂酰乙醇胺（PE）的百分比并降低了磷脂酰胆碱（PC），从而导致PC/PE比率降低。结果表明，BTN1A1 通过涉及膜磷脂组成的机制在调节 LD 合成中起重要作用。