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

分离了秀丽线虫磷脂酶D(PLD-1)基因。该基因编码1,427个氨基酸的蛋白质，包含4个由细菌、植物、酵母和哺乳动物PLD的一级结构定义的保守区。此外，对PLD活性至关重要的II和IV区域的HKD基序(HxKxxxxD)是完全保守的。相应的基因组序列被粘粒克隆C04G6所覆盖。C04G6与克隆的cDNA序列比较表明，PLD基因有18个外显子，转录单位为7.8kb。用BLAST算法对线虫全基因组进行的广泛筛选表明，PLD-1是PLD家族中唯一的成员，该家族包含四个保守区，其中包括两个催化HKD基序。在COS-7细胞中表达的蛋白在0.3%丁醇存在下催化转磷脂酰化反应生成磷脂酰丁醇。它还能从磷脂酰胆碱中释放出[3 H]胆碱，表明表达的蛋白具有PLD活性。这种PLD活性依赖于磷脂酰肌醇4，5-二磷酸，并受到ADP-核糖化因子的弱刺激。而油酸对其有较强的抑制作用。这些特性与哺乳动物的PLD2相似。为了确定PLD-1在哪里表达，我们构建了PLD-1与绿色荧光蛋白(GFP)之间的翻译融合，以产生PLD-1：：GFP。该基因除含有668个氨基酸外，还含有约4.7kb的PLD-1启动子区。PLD-1在咽肌和大部分神经元中有表达。上皮细胞、体壁肌肉、外阴肌和排泄管也有弱表达。

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.