Posttranslational modification of glycosylphosphatidylinositol (GPI)-specific phospholipase D and its activity in cleavage of GPI anchors.
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.
Tsujioka
H
H
Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka, Jonan-ku, 814-0180, Japan.
Misumi
Y
Y
Takami
N
N
Ikehara
Y
Y
Tujioka
H
H
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D015723:Gene Library; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution
D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; C055791:germ-cell AP isoenzyme; D000469:Alkaline Phosphatase; D010739:Phospholipase D; C065017:glycoprotein phospholipase D