Abstract
Glycosylphosphatidylinositols (GPIs) are linked to many cell-surface proteins, anchor these proteins in the membrane, and are well characterized. However, GPIs that exist in the free form on the mammalian cell surface remain largely unexplored. To investigate free GPIs in cultured cell lines and mouse tissues, here we used the T5-4E10 mAb (T5 mAb), which recognizes unlinked GPIs having an N-acetylgalactosamine (GalNAc) side chain linked to the first mannose at the nonreducing terminus. We detected free GPIs bearing the GalNAc side chain on the surface of Neuro2a and CHO, but not of HEK293, K562, and C2C12 cells. Furthermore, free GPIs were present in mouse pons, medulla oblongata, spinal cord, testis, epididymis, and kidney. Using a panel of Chinese hamster ovary cells defective in both GPI-transamidase and GPI remodeling pathway, we demonstrate that free GPIs follow the same structural remodeling pathway during passage from the endoplasmic reticulum to the plasma membrane as do protein-linked GPI. Specifically, free GPIs underwent post-GPI attachment to protein 1 (PGAP1)-mediated inositol deacylation, PGAP5-mediated removal of the ethanolamine phosphate from the second mannose, and PGAP3- and PGAP2-mediated fatty acid remodeling. Moreover, T5 mAb recognized free GPIs even if the inositol-linked acyl chain or ethanolamine-phosphate side chain linked to the second mannose is not removed. In contrast, addition of a fourth mannose by phosphatidylinositol glycan anchor biosynthesis class Z (PIGZ) inhibited T5 mAb-mediated detection of free GPIs. Our results indicate that free GPIs are normal components of the plasma membrane in some tissues and further characterize free GPIs in mammalian cells.
Highlights
Glycosylphosphatidylinositols (GPIs) are linked to many cellsurface proteins, anchor these proteins in the membrane, and are well characterized
Using T5-4E10 mAb (T5 mAb) that recognizes free GPI bearing a Man1-linked GalNAc side chain, we show its presence on the cell surface of some cultured cell lines by flow cytometry and in the lysate of mouse pons, medulla oblongata, spinal cord, kidney, testis, and epididymis by Western blotting
Taking advantage of various mutant Chinese hamster ovary (CHO) cells defective in GPI maturation and based on the binding specificity of T5 mAb, known structures of protein GPI-anchors and sensitivity to phosphatidylinositol-specific phospholipase C (PI-PLC), we conclude that free GPIs take basically the same maturation pathway as GPI-anchored proteins (GPI-APs)
Summary
Addition of a fourth mannose by phosphatidylinositol glycan anchor biosynthesis class Z (PIGZ) inhibited T5 mAb-mediated detection of free GPIs. Our results indicate that free GPIs are normal components of the plasma membrane in some tissues and further characterize free GPIs in mammalian cells. It was shown that T5 mAb recnese hamster ovary; ER, endoplasmic reticulum; GPI-Tase, GPI transamidase; PNH, paroxysmal nocturnal hemoglobinuria; EtNP, ethanolamine phosphate; PI-PLC, phosphatidylinositol-specific phospholipase C; KO, knockout; PVDF, polyvinylidene difluoride; TfR, transferrin receptor; PIGZ, phosphatidylinositol glycan anchor biosynthesis class Z; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Our results indicate that free GPIs undergo similar structural remodeling to GPI-APs
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
