Properties of exogenously added GPI-anchored proteins following their incorporation into cells

✍ Scribed by Daniel R.D. Premkumar; Yoshihiro Fukuoka; Daniel Sevlever; Elaine Brunschwig; Terrone L. Rosenberry; Mark L. Tykocinski; M. Edward Medof

• Publisher: John Wiley and Sons
• Year: 2001
• Tongue: English
• Weight: 234 KB
• Volume: 82
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.1154

✦ Synopsis

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. J. Cell. Biochem. 82: 234–245, 2001. © 2001 Wiley‐Liss, Inc.