title: Implications of lipid moiety in oligomerization and immunoreactivities of GPI-anchored proteins.
abstract: Glycosylphosphatidylinositol (GPI) enriches GPI-anchored proteins (GPI-AP) in lipid rafts by intimate interaction of its lipid moiety with sphingolipids and cholesterol. In addition to such lipid-lipid interactions, it has been reported that GPI may interact with protein moiety linked to GPI and affect protein conformations because GPI delipidation reduced immunoreactivities of protein. Here, we report that GPI-APs that have not undergone fatty acid remodeling exhibit reduced immunoreactivities in Western blotting, similar to delipidated proteins, compared with normal remodeled GPI-APs. In contrast, immunostaining in flow cytometry and immunoprecipitation did not show significant differences between remodeled and unremodeled GPI-APs. Moreover, detection with premixed primary/secondary antibody complexes or Fab fragments eliminated this difference in Western blotting. These results indicate that normally remodeled GPI enhanced oligomerization of GPI-APs and that inefficient oligomerization of unremodeled GPI-APs was responsible for reduced immunoreactivities. Moreover, the reduction in immunoreactivities of delipidated GPI-APs was most likely caused by the same effect. Finally, by chemical cross-linking of surface proteins in living cells and cell killing assay using a pore-forming bacterial toxin, we showed that enhanced oligomerization by GPI-remodeling occurs under a physiological membrane environment. Thus, this study clarifies the significance of GPI fatty acid remodeling in oligomerization of GPI-APs and provides useful information for technical studies of these cell components.
authors: Department of Immunoregulation, Research Institute for Microbial Diseases, and Laboratory of Immunoglycobiology, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan.
authors: Jihyoun
authors: J
authors: Seong
authors: Yetao
authors: Y
authors: Wang
authors: Taroh
authors: T
authors: Kinoshita
authors: Yusuke
authors: Y
authors: Maeda
mesh_terms: D000818:Animals; D058851:GPI-Linked Proteins / Q000737:chemistry* / Q000378:metabolism*; D047468:Immunoprecipitation; D021962:Membrane Microdomains; D051379:Mice; D018345:Mice, Knockout; D055503:Protein Multimerization
chemical_list: D058851:GPI-Linked Proteins
references: Neuron. 2000 Mar;25(3):611-23
references: 10774729
references: J Biol Chem. 2004 Apr 2;279(14):14256-63
references: 14734546
references: J Biol Chem. 2012 Feb 24;287(9):6318-25
references: 22228761
references: J Biol Chem. 1997 Sep 5;272(36):22781-7
references: 9278439
references: Mol Biol Cell. 2006 Mar;17(3):1410-20
references: 16407401
references: Biochem Biophys Res Commun. 2012 Jan 27;417(4):1235-41
references: 22227195
references: J Biol Chem. 2007 Oct 19;282(42):30373-80
references: 17711852
references: J Lipid Res. 2007 May;48(5):993-1011
references: 17361015
references: Trends Microbiol. 2000 Apr;8(4):168-72
references: 10754575
references: Mol Biol Cell. 2007 Apr;18(4):1497-506
references: 17314402
references: FASEB J. 2001 Feb;15(2):545-8
references: 11156970
references: Cell. 2009 Oct 16;139(2):352-65
references: 19837036
references: J Cell Biol. 2011 Jul 11;194(1):61-75
references: 21727194
references: Nat Chem Biol. 2012 Sep;8(9):774-83
references: 22820419
references: J Cell Biol. 2004 Nov 22;167(4):699-709
references: 15557121
references: J Cell Sci. 1995 Feb;108 ( Pt 2):487-97
references: 7539435
references: Cell. 1992 Feb 7;68(3):533-44
references: 1531449
references: Int J Cancer Suppl. 1988;2:50-8
references: 2450848
references: Nat Methods. 2011 Sep 18;8(11):969-75
references: 21926998
references: Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12130-4
references: 7991596
references: Nat Rev Mol Cell Biol. 2000 Oct;1(1):31-9
references: 11413487
references: J Immunol. 1999 May 15;162(10):5993-7
references: 10229838