Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis

Squalene epoxidase, a membrane-associated enzyme that converts squalene to squalene 2,3-oxide, plays an important role in the maintenance of cholesterol homeostasis. In 1957, Bloch and colleagues identified a factor from rat liver cytosol termed “supernatant protein factor (SPF),” which promotes the squalene epoxidation catalyzed by rat liver microsomes with oxygen, NADPH, FAD, and phospholipid [Tchen, T. T. & Bloch, K. (1957) J. Biol. Chem. 226, 921–930]. Although purification of SPF by 11,000-fold was reported, no information is so far available on the primary structure or biological function of SPF. Here we report the cDNA cloning and expression of SPF from rat and human. The encoded protein of 403 amino acids belongs to a family of cytosolic lipid-binding/transfer proteins such as α-tocopherol transfer protein, cellular retinal binding protein, yeast phosphatidylinositol transfer protein (Sec14p), and squid retinal binding protein. Recombinant SPF produced in Escherichia coli enhances microsomal squalene epoxidase activity and promotes intermembrane transfer of squalene in vitro. SPF mRNA is expressed abundantly in the liver and small intestine, both of which are important sites of cholesterol biosynthesis. SPF is expressed significantly in isolated hepatocytes, but the expression level was markedly decreased after 48 h of in vitro culture. Moreover, SPF was not detectable in most of the cell lines tested, including HepG2 and McARH7777 hepatomas. Transfection of SPF cDNA in McARH7777 significantly stimulated de novo cholesterol biosynthesis. These data suggest that SPF is a cytosolic squalene transfer protein capable of regulating cholesterol biosynthesis.

鲨烯环氧酶（squalene epoxidase）是一种膜结合酶，可将鲨烯转化为鲨烯2,3-环氧化物，在维持胆固醇稳态（cholesterol homeostasis）中发挥关键作用。1957年，Bloch及其同事从大鼠肝胞液中鉴定出一种被命名为“上清液蛋白因子（supernatant protein factor，SPF）”的因子，该因子可在氧气、烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、黄素腺嘌呤二核苷酸（FAD）及磷脂存在的条件下，促进大鼠肝微粒体催化的鲨烯环氧反应[Tchen, T. T. & Bloch, K. (1957) J. Biol. Chem. 226, 921–930]。尽管已有研究将SPF纯化了11000倍，但截至目前，尚无关于SPF一级结构或生物学功能的相关报道。

本研究报道了大鼠和人源SPF的cDNA克隆与表达。该基因编码的蛋白含403个氨基酸，属于胞质脂质结合/转运蛋白家族，该家族涵盖α-生育酚转运蛋白、细胞视黄醇结合蛋白、酵母磷脂酰肌醇转运蛋白（Sec14p）以及鱿鱼视黄醇结合蛋白。在大肠杆菌中重组表达的SPF可在体外增强微粒体鲨烯环氧酶活性，并促进鲨烯的膜间转运。

SPF mRNA在肝脏和小肠中呈高表达，而这两种组织均为胆固醇生物合成的关键位点。在分离的原代肝细胞中SPF表达显著，但经体外培养48小时后，其表达水平显著下调。此外，在绝大多数检测的细胞系中均未检测到SPF，包括HepG2和McARH7777肝癌细胞。将SPF cDNA转染至McARH7777细胞后，可显著刺激胆固醇的从头生物合成。

上述研究结果提示，SPF是一种能够调控胆固醇生物合成的胞质鲨烯转运蛋白。