Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase
Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase
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DOI：
10.1042/0264-6021:3400561
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发表时间：
1999-06-01
影响因子：
4.1
通讯作者：
Subramani, S
中科院分区：
文献类型：
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作者：
Fransen, M;Van Veldhoven, PP;Subramani, S
为了阐明未知的哺乳动物过氧化物酶体酶和功能，我们使用针对过氧化物酶体亚组分的抗体，对编码蛋白VI的基因与大鼠肝脏cDNA文库之间表达融合的M13噬菌体进行体外免疫亲和选择（生物筛选）。在最初的一系列生物筛选实验中，获得了四个不同的cDNA克隆。这些cDNA物种编码两种先前鉴定的过氧化物酶体酶，过氧化氢酶和尿酸氧化酶，以及两种含有c端过氧化物酶体靶向信号（PTS1）的新蛋白。对这些新蛋白的初步结构分析表明，其中一个以三肽AKL结尾，与酵母过氧化物酶体2,4-二烯酰辅酶A还原酶（EC 1.3.1.34； DCR）同源，后者是降解不饱和脂肪酸所需的酶；另一个以三肽SRL结尾，是短链脱氢酶/还原酶（SDR）家族的成员，有三个同工型。编码GFP- dcr - akl、GFP- dcr、GFP- sdr和GFP- sdr的绿色荧光蛋白（GFP）融合物在哺乳动物细胞中表达。重组融合蛋白的亚细胞定位分析证实了GFP-DCR-AKL和GFP-SDR-SRL的过氧化物酶体定位，以及PTS1的功能。通过显示细菌表达蛋白的DCR活性，证实了AKL蛋白确实是nadph依赖性的DCR。这些结果在分子水平上证明哺乳动物过氧化物酶体确实含有DCR。此外，本文的结果表明，蛋白质VI展示系统适用于cDNA文库中罕见cDNA克隆的分离，该技术有助于鉴定新的过氧化物酶体蛋白。
To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions. In an initial series of biopanning experiments, four different cDNA clones were obtained. These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1). A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms. Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells. The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1, That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein. These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR. In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins.