Gossett R E, Edmondson R D, Jolly C A, Cho T H, Russell D H, Knudsen J, Kier A B, Schroeder F
Department of Veterinary Pathobiology, Texas A & M University, TVMC, College Station, Texas 77843-4467, USA.
Arch Biochem Biophys. 1998 Feb 15;350(2):201-13. doi: 10.1006/abbi.1997.0521.
Acyl-CoA binding protein (ACBP) is a ubiquitous cytosolic protein found in high levels in tumorigenic cells. However, the molecular basis for the elevated levels of ACBP in malignant cells, ligand binding characteristics, and function in microsomal phospholipid synthesis have not been resolved. To address whether tumorigenic ACBP differs from the native protein, ACBP was purified from LM cells, a tumorigenic subline of mouse L-929 fibroblasts, and its primary structure was examined by delayed-extraction MALDI-linear TOF mass spectrometry. Proteolytic digestion and peptide sequence analysis confirmed that ACBP from LM cells was identical to native mouse ACBP (based on cDNA-derived amino acid sequence) with no amino acid substitutions, deletions, or posttranslational modifications. A fluorescent binding assay revealed that mouse ACBP bound cis-parinaroyl-CoA with high affinity, Kd 7.6 +/- 2.3 nM, at a single binding site. Furthermore, mouse ACBP enhanced microsomal phosphatidic acid formation from oleoyl-CoA 2.3-fold. Mouse ACBP also inhibited microsomal phospholipid acyl chain remodeling of choline-containing phospholipids, phosphatidylcholine and sphingomyelin, by 50 and 64%, respectively. These effects were specific compared to those of native rat liver or recombinant rat ACBP. Mouse and rat ACBPs differed by three amino acid substitutions at positions 4, 68, and 78. Although these small differences in amino acid sequence did not alter binding affinity for cis-parinaroyl-CoA, rat liver ACBP stimulated utilization of oleoyl-CoA 3.8-fold by microsomal glycerol-3-phosphate acyltransferase, significantly higher than that observed with mouse ACBP, but did not alter microsomal phospholipid acyl chain remodeling from oleoyl-CoA. In addition, these ACBPs protected oleoyl-CoA against hydrolysis. Finally, both mouse and rat ACBP shifted the incorporation of oleoyl-CoA from microsomal phospholipid acyl chain remodeling to phosphatidic acid biosynthesis. These data for the first time show a role for ACBP in stimulating microsomal phosphatidic acid biosynthesis and acyl chain remodeling in vitro. While ACBP from tumorigenic cells did not differ from normal, ACBPs from different murine species displayed subtle differences in their effects on microsomal phospholipid metabolism in vitro.
酰基辅酶A结合蛋白（ACBP）是一种普遍存在的胞质蛋白，在致瘤细胞中含量很高。然而，恶性细胞中ACBP水平升高的分子基础、配体结合特性以及在微粒体磷脂合成中的功能尚未明确。为了研究致瘤性ACBP是否与天然蛋白不同，从LM细胞（小鼠L-929成纤维细胞的一个致瘤亚系）中纯化了ACBP，并通过延迟提取基质辅助激光解吸电离线性飞行时间质谱法检测其一级结构。蛋白水解消化和肽序列分析证实，LM细胞中的ACBP与天然小鼠ACBP（基于cDNA推导的氨基酸序列）相同，没有氨基酸替换、缺失或翻译后修饰。荧光结合试验表明，小鼠ACBP在单个结合位点以高亲和力（Kd 7.6±2.3 nM）结合顺式-杷荏油酰辅酶A。此外，小鼠ACBP使油酰辅酶A生成微粒体磷脂酸的能力增强了2.3倍。小鼠ACBP还分别抑制了微粒体中含胆碱磷脂（磷脂酰胆碱和鞘磷脂）的磷脂酰基链重塑50%和64%。与天然大鼠肝脏或重组大鼠ACBP相比，这些作用具有特异性。小鼠和大鼠的ACBP在第4、68和78位有三个氨基酸替换。尽管这些氨基酸序列上的微小差异并未改变对顺式-杷荏油酰辅酶A的结合亲和力，但大鼠肝脏ACBP能使微粒体甘油-3-磷酸酰基转移酶利用油酰辅酶A的能力提高3.8倍，显著高于小鼠ACBP，但并未改变油酰辅酶A引起的微粒体磷脂酰基链重塑。此外，这些ACBP能保护油酰辅酶A不被水解。最后，小鼠和大鼠ACBP都将油酰辅酶A从微粒体磷脂酰基链重塑的掺入转移到磷脂酸生物合成中。这些数据首次表明ACBP在体外刺激微粒体磷脂酸生物合成和酰基链重塑中发挥作用。虽然致瘤细胞中的ACBP与正常细胞中的没有差异，但来自不同鼠种的ACBP在体外对微粒体磷脂代谢的影响上存在细微差异。
