Functional analysis of phospholipase D (PLD) superfamily in Caenorhabditis elegans

秀丽隐杆线虫磷脂酶 D (PLD) 超家族的功能分析

基本信息

批准号：
12680689
负责人：
NAKASHIMA Shigeru
金额：
$ 0.9万
依托单位：
Gifu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680689/
关键词：
Caenorhabditis elegans phospholipase D pld-1 gene cDNA phospholipase D superfamily cell-specific expression gene knockout GFP SL-1

项目摘要

The gene encoding phospholipase D (PLD) of Caenorhabditis elegans (pld-1) was isolated. The isolated cDNA encodes a 1,427 amino acid protein which contains four conserved regions defined by the primary structures of bacterial, plant, yeast and mammalian PLDs. Furthermore, HKD motifs (HxKxxxxD) in regions II and IV, which are critical for PLD activity, are completely conserved. The corresponding genomic sequence was covered by the cosmid clone C04G6. Comparison of the sequences between C04G6 and the cloned cDNA revealed that PLD gene has 18 exons and that the transcription unit is 7.8 kb. An extensive screening of the complete C. elegans genome by the BLAST algorithm has revealed that PLD-1 is the sole member of the PLD family which contains four conserved regions including two catalytic HKD motifs. The protein expressed in COS-7 cells catalyzed transphosphatidylation reaction to generate phosphatidylbutanol in the presence of 0.3 % butanol. It also released [^3H]choline from [^3H]phosphatidylcholine, indicating that expressed protein exhibited PLD activity. This PLD activity was dependent on phosphatidylinositol 4,5-bisphosphate and weakly stimulated by ADP-ribosylation factor. However, it was strongly inhibited by oleic acid. These properties are similar to those of mammalian PLD2. To determine where pld-1 is expressed, we constructed a translational fusion between pld-1 and the green fluorescent protein (GFP) to generate pld-1::GFP. This construct contains about 4.7 kb pld-1 promoter regions in addition to first 668 amino acids. PLD-1 was expressed in pharyngeal muscles and most of neurons. Weak expression was also observed in epithelial cells, body-wall muscles, vulval muscles and excretory canal.

分离了编码秀丽隐杆线虫磷脂酶 D (PLD) 的基因 (pld-1)。分离的 cDNA 编码 1,427 个氨基酸的蛋白质，其中包含由细菌、植物、酵母和哺乳动物 PLD 的一级结构定义的四个保守区域。此外，对于 PLD 活性至关重要的 II 区和 IV 区中的 HKD 基序 (HxKxxxxD) 是完全保守的。相应的基因组序列被粘粒克隆C04G6覆盖。将C04G6与克隆的cDNA序列进行比较，发现PLD基因有18个外显子，转录单位为7.8 kb。通过 BLAST 算法对完整的线虫基因组进行的广泛筛选表明，PLD-1 是 PLD 家族的唯一成员，该家族包含四个保守区域，其中包括两个催化 HKD 基序。 COS-7细胞中表达的蛋白质在0.3%丁醇存在下催化转磷脂酰化反应生成磷脂酰丁醇。它还从[^3H]磷脂酰胆碱中释放[^3H]胆碱，表明表达的蛋白质表现出PLD活性。该 PLD 活性依赖于磷脂酰肌醇 4,5-二磷酸，并受 ADP-核糖基化因子微弱刺激。然而，它受到油酸的强烈抑制。这些特性与哺乳动物 PLD2 相似。为了确定 pld-1 的表达位置，我们构建了 pld-1 和绿色荧光蛋白 (GFP) 之间的翻译融合，以生成 pld-1::GFP。除了前 668 个氨基酸之外，该构建体还包含约 4.7 kb pld-1 启动子区域。 PLD-1在咽部肌肉和大部分神经元中表达。在上皮细胞、体壁肌肉、外阴肌肉和排泄道中也观察到弱表达。