TY - JOUR
T1 - Properties of exogenously added GPI-anchored proteins following their incorporation into cells
AU - Premkumar, Daniel R.D.
AU - Fukuoka, Yoshihiro
AU - Sevlever, Daniel
AU - Brunschwig, Elaine
AU - Rosenberry, Terrone L.
AU - Tykocinski, Mark L.
AU - Edward Medof, M.
PY - 2001
Y1 - 2001
N2 - 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 (Ehu) decay accelerating factor, Ehu 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; nonphysiological attachment and/or incomplete insertion, uncomplexed membrane integration, and 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.
AB - 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 (Ehu) decay accelerating factor, Ehu 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; nonphysiological attachment and/or incomplete insertion, uncomplexed membrane integration, and 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.
KW - GPI-anchored protein
KW - Incorporation
KW - Microdomain
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U2 - 10.1002/jcb.1154
DO - 10.1002/jcb.1154
M3 - Article
C2 - 11527149
AN - SCOPUS:0034960693
SN - 0730-2312
VL - 82
SP - 234
EP - 245
JO - Journal of cellular biochemistry
JF - Journal of cellular biochemistry
IS - 2
ER -