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Properties of exogenously added GPI-anchored proteins following their incorporation into cells.
- Source :
-
Journal of cellular biochemistry [J Cell Biochem] 2001; Vol. 82 (2), pp. 234-45. - Publication Year :
- 2001
-
Abstract
- Isolated glycosylphosphatidylinositol (GPI)-anchored proteins, when added to cells in vitro, incorporate into their surface membranes and, once incorporated, exert their native functions. Virtually any protein of interest, if expressed as a GPI-reanchored derivative, can be modified to acquire this capacity. Such transfer of proteins directly to cells, termed "protein engineering" or "painting" constitutes an alternative to conventional gene transfer for manipulating cell surface composition that has many potential applications. Previous studies with incorporated GPI-anchored proteins have focused almost entirely on their extracellular functions. In this study, biotinylated human erythrocyte (E(hu)) decay accelerating factor, E(hu) acetylcholinesterase, and GPI-reanchored murine B7-1 and B7-2 were used as GPI-anchored reporters to characterize their plasma membrane organization and cell signalling properties following addition to Hela or Chinese hamster ovary cells. For each reporter, three types of cell-association were documented; (1) nonphysiological attachment and/or incomplete insertion, (2) uncomplexed membrane integration, and (3) organization into TX-100-resistant microdomains. Transit from the first two compartments into the third, i.e., microdomains, progressed slowly, continuing even after 24 to 36 h and was associated with the acquisition of cell signalling capacity. All four reporters, incorporated in two different detergents, behaved similarly. When organized in microdomains, caveolin and other GPI proteins co-isolated with the incorporated reporter. These results have implications for protein engineering of cells in general, and in particular, for cells such as modified tumor cell immunogens administered to patients for therapeutic purposes.
- Subjects :
- Acetylcholinesterase metabolism
Animals
Antigens, CD metabolism
B7-1 Antigen metabolism
B7-2 Antigen
Biotinylation
CD55 Antigens metabolism
CHO Cells
Cancer Vaccines
Cell Compartmentation
Centrifugation, Density Gradient
Cricetinae
Cricetulus
Electrophoresis, Polyacrylamide Gel
Glycosylphosphatidylinositols administration & dosage
Glycosylphosphatidylinositols metabolism
HeLa Cells
Humans
Membrane Glycoproteins metabolism
Membrane Microdomains metabolism
Mice
Phosphorylation
Protein Engineering
Protein Processing, Post-Translational
Recombinant Fusion Proteins metabolism
Time Factors
Transfection
Cell Membrane metabolism
Glycosylphosphatidylinositols pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 0730-2312
- Volume :
- 82
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of cellular biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 11527149
- Full Text :
- https://doi.org/10.1002/jcb.1154