Kamisaka Yasushi, Goto Rie, Shibakami Motonari, Yoshioka Kyoko, Sato Yukari
National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Biosci Biotechnol Biochem. 2011;75(6):1135-9. doi: 10.1271/bbb.110034. Epub 2011 Jun 13.
To measure the interactions of diacylglycerol acyltransferase (DGAT) by surface plasmon resonance (SPR), we immobilized Saccharomyces cerevisiae DGAT2 encoded by DGA1 on a BIACORE sensor chip surface. We used N-terminally truncated Dga1p with a FLAG tag at the C-terminus, which was purified to apparent homogeneity, maintaining significant DGAT activity (Kamisaka et al., Appl. Microbiol. Biotechnol., 88, 105-115 (2010)). Truncated Dga1p with a FLAG tag was immobilized with an anti-FLAG antibody that had been coupled with an L1 chip surface consisting of a carboxymethyl dextran matrix with additional hydrophobic alkane groups. The Dga1p-immobilized chip surface was analyzed for interactions of Dga1p with oleoyl-CoA, its substrate, and anti-Dga1p IgG, its interacting protein, by SPR. The binding of these analytes with the Dga1p-immobilized chip surface was specific, because butyryl-CoA, which cannot be used as a substrate for DGAT, and anti-glyceraldehyde-3-phosphate dehydrogenase IgG, did not induce any signals on SPR. Furthermore, injection of organic compounds such as xanthohumol, a DGAT inhibitor, into the Dga1p-immobilized chip surface induced significant SPR signals, probably due to interaction with DGAT. Another DGAT inhibitor, piperine, did not induce SPR signals on application, but induced them due to piperine on application together with oleoyl-CoA, in which piperine can be incorporated into the micelles of oleoyl-CoA. The results indicate that the Dga1p-immobilized L1 chip surface recognized DGAT inhibitors. Taking all this together, SPR measurement using the Dga1p-immobilized L1 chip surface provided a useful system to elucidate the structure-function relationships of DGAT and screen DGAT inhibitors.
为了通过表面等离子体共振（SPR）测量二酰基甘油酰基转移酶（DGAT）的相互作用，我们将由DGA1编码的酿酒酵母DGAT2固定在BIACORE传感器芯片表面。我们使用了在C端带有FLAG标签的N端截短的Dga1p，其被纯化至表观均一，保持了显著的DGAT活性（Kamisaka等人，《应用微生物学与生物技术》，88，105 - 115（2010））。带有FLAG标签的截短Dga1p用已与由具有额外疏水烷烃基团的羧甲基葡聚糖基质组成的L1芯片表面偶联的抗FLAG抗体固定。通过SPR分析固定有Dga1p的芯片表面上Dga1p与它的底物油酰辅酶A以及它的相互作用蛋白抗Dga1p IgG的相互作用。这些分析物与固定有Dga1p的芯片表面的结合是特异性的，因为不能用作DGAT底物的丁酰辅酶A以及抗甘油醛 - 3 - 磷酸脱氢酶IgG在SPR上未诱导任何信号。此外，将诸如DGAT抑制剂黄腐酚之类的有机化合物注入固定有Dga1p的芯片表面会诱导显著的SPR信号，这可能是由于与DGAT的相互作用。另一种DGAT抑制剂胡椒碱在应用时未诱导SPR信号，但在与油酰辅酶A一起应用时会诱导信号，因为胡椒碱可以掺入油酰辅酶A的胶束中。结果表明固定有Dga1p的L1芯片表面能够识别DGAT抑制剂。综合所有这些，使用固定有Dga1p的L1芯片表面进行SPR测量提供了一个有用的系统来阐明DGAT的结构 - 功能关系并筛选DGAT抑制剂。
