Tsujioka H, Misumi Y, Takami N, Ikehara Y
Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka, Jonan-ku, 814-0180, Japan.
Biochem Biophys Res Commun. 1998 Oct 29;251(3):737-43. doi: 10.1006/bbrc.1998.9542.
Human glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was exogenously expressed in mammalian CHO cells and in insect H5 cells. GPI-PLD was initially synthesized as a 105-kDa form and then secreted as a mature 115-kDa form from the CHO cells, whereas it was secreted as an immature 98-kDa form from the H5 cells. The difference of the molecular forms was caused by its oligosaccharide processing in the two cell lines. These forms showed a different reactivity to anti-C-terminal peptide of GPI-PLD; the 105-kDa and 98-kDa forms were directly recognized by the antibodies, whereas the 115-kDa form was immunoreactive only after being denatured. In an in vitro assay, the 98-kDa form but not the 115-kDa form was able to release a significant amount of GPI-anchored proteins from intact membranes, although the two forms had the same GPI-anchor cleavage activity in the presence of detergents. In addition, a GPI-anchored protein, when coexpressed in CHO cells, was intracellularly cleaved by GPI-PLD in the secretory pathway. Taken together, these results suggest that GPI-PLD undergoes a conformational change by posttranslational modification, which affects its immunoreactive and enzymatic properties.
人糖基磷脂酰肌醇特异性磷脂酶D（GPI-PLD）在哺乳动物CHO细胞和昆虫H5细胞中进行外源表达。GPI-PLD最初以105 kDa的形式合成，然后从CHO细胞中作为成熟的115 kDa形式分泌，而从H5细胞中则作为未成熟的98 kDa形式分泌。分子形式的差异是由其在两种细胞系中的寡糖加工引起的。这些形式对GPI-PLD的抗C末端肽表现出不同的反应性；105 kDa和98 kDa的形式可被抗体直接识别，而115 kDa的形式仅在变性后具有免疫反应性。在体外试验中，98 kDa的形式而非115 kDa的形式能够从完整膜中释放大量的GPI锚定蛋白，尽管这两种形式在存在去污剂的情况下具有相同的GPI锚定切割活性。此外，一种GPI锚定蛋白在CHO细胞中共表达时，在分泌途径中被GPI-PLD在细胞内切割。综上所述，这些结果表明GPI-PLD通过翻译后修饰发生构象变化，这影响了其免疫反应性和酶活性。
