人骨髓基质细胞中糖基化磷脂酰肌醇特异性磷脂酶DcDNA的克隆

Cloning of Glycosylphosphatidylinositol-Specific Phospholipase D cDNA from Human Bone Marrow Stromal Cells

为探讨人糖基化磷脂酰肌醇特异性磷脂酶D(GPI PLD)cDNA的结构及功能 ,应用RT PCR从人骨髓基质细胞中克隆了长约 2 6kb的GPI PLDcDNA ,包含完整阅读框架 ,编码 2 3个氨基酸的信号肽及 817个氨基酸的成熟肽 .该cDNA与人胰腺GPI PLDcDNA几乎百分之百同源 ,与人肝脏GPI PLDcDNA同源性为 95 %,氨基酸同源性为 94 %,3者对应的结构基因只有 1个 ,位于人类第 6号染色体上 ,基因组序列长约 80kb ,包括 2 5个外显子 .构建克隆的GPI PLDcDNA的真核表达载体 ,通过脂质体转染能表达GPI锚定的胎盘型碱性磷酸酶 (PLAP)而无GPI PLD活性的G9细胞 ,同时设立对照组检测GPI PLDcDNA的功能 .结果显示 ,对照组细胞几乎检测不到GPI PLD活性 ,其表达的PLAP主要位于细胞膜上 ;而转染GPI PLDcDNA的G9细胞能检测到较高水平的GPI PLD活性 ,而且大部分酶活性存在于培养液中 ,其表达的PLAP也主要被释放入培养液 .结果证实 ,从人骨髓基质细胞中克隆的GPI PLDcDNA有生物学功能 ,它能释放细胞膜上GPI锚定蛋白质 .

To explore the structure and function of human glycosylphosphatidylinositol-specific phospholipase D(GPI-PLD) cDNA, a GPI-PLD cDNA with the size of approximately 2.6 kb was cloned from human bone marrow stromal cells using RT-PCR. This target gene had a complete open reading frame coding a signal peptide of 23 amino acids and a mature peptide of 817 amino acids. Its nucleotide sequence analysis showed nearly 100% identity of nucleotide sequence to the reported human pancreatic sequence, and 95% identity of nucleotide sequence to the reported human liver sequence. Analysis of deduced amino acid sequence showed 94% identity of amino acid sequence to the reported human liver sequence. Moreover, the three human GPI-PLD cDNA sequences represented products of the same genomic gene which covered about 80 kb on chromosome 6.The comparative analysis showed that the genomic gene encoding GPI-PLD comprised of 25 exons. To observe the function of GPI-PLD cDNA, the cloned gene was ligated into the eukaryotic expression plasmid, pcDNA3.1(+), and the resulting plasmid, pcDNA3.1(+)/GPI-PLD, was introduced into G 9 cells which contained GPI-anchored PLAP but no GPI-PLD activity by liposome transfection. The results showed that nearly no GPI-PLD activity was detected in control groups whose PLAP activity was mainly distributed in the cell pellets, whereas high levels of GPI-PLD activity were discovered in the cells transfected with pcDNA3.1(+)/GPI-PLD. Furthermore, the majority of GPI-PLD activity was secreted into the culture supernatants with the remainder being associated with the cell pellets. A significant increase was observed in the amount of PLAP activity released into culture supernatants. The results suggested that the cloned GPI-PLD cDNA was biologically active which could release GPI-anchored proteins on the cell surface.