Cell‐surface engineering with GPI‐anchored proteins

Abstract

Protein engineering of cell surfaces is a potentially powerful technology through which the surface protein composition of cells can be manipulated without gene transfer. This technology exploits the fact that proteins that are anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, incorporate into their surface membranes and are fully functional. By substituting 3'-mRNA end sequence of naturally GPI-anchored proteins (i.e., a sequence that contains the signals that direct GPI anchoring) for endogenous 3'-mRNA end sequence, virtually any protein of interest can be expressed as a GPI-anchored derivative. The GPI-anchored product then can be purified from transfectants and the purified protein used to "paint" any target cell. Such protein engineering or "painting" of the cell surface offers several advantages over conventional gene transfer. Among these advantages are that 1) GPI-anchored proteins can be painted onto cells that are difficult to transfect, 2) cells can be altered immediately without previous culturing, 3) the amount of protein added to the surface can be precisely controlled, and 4) multiple GPI-anchored proteins can be sequentially or concurrently inserted into the same cells. Emerging applications for the technology include its use for the analysis of complex cell-surface interactions, the engineering of antigen presenting cells, the development of cancer vaccines, and possibly the protection against graft rejection.

Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells

“…Glycosylphosphatidylinositol (GPI) membrane anchors are found on many eukaryotic cell-surface glycoproteins, and their structures, functions and biosynthesis have been extensively reviewed [1][2][3][4][5][6]. The minimum GPI-anchor structure is NH # CH # CH # -HPO % -6Manα1-2Manα1-6Manα1-4GlcNα1-6-myo-inositol-1-HPO % -lipid (EtNP-Man $ GlcN-PI), where the lipid may be diacylglycerol, alkylacylglycerol or ceramide [1].…”

“…The biosynthesis of GPI anchors involves the addition of GlcNAc to phosphatidylinositol (PI), to give GlcNAc-PI, which is de-N-acetylated to form GlcN-PI [6][7][8][9]. A Chinese hamster ovary cell mutant that is deficient in GlcNAc-PI de-N-acetylase activity has been described recently [10] but the defective gene has yet to be isolated.…”

The α vβ 3 integrin as a tumor homing ligand for lymphocytes

“…Studies in humans with parasitic infections or mice transgenic for DAF had already provided evidence that insertion of dissociated GPIanchored proteins into cell membranes occurs in vivo [42,43]. The addition of a glypiation signal to cDNA permits expression of the protein as a GPI-anchored form [30]. In our hands, production of the GPI fusion protein was optimal in fast-growing HEK293 cells enabling purification of large quantities of KISS31-GPI from cell lysates.…”

“…To confirm that the newly generated molecules were indeed inserted into the plasma membrane of the cell via a GPI anchor, we incubated the cells with phosphatidylinositol-specific phospholipase C ( Fig. 2A), which specifically cleaves most GPI-linked proteins [30,37], including KISS31-GPI and CD31-GPI.…”

“…Therefore, we have applied a novel strategy to insert § v g 3 integrin ligands into primary lymphocytes without the need for genetic manipulation of the cells. This technology, termed cell surface 'painting', makes use of the unusual capacity of purified glycosylphosphatidylinositol (GPI)-anchored (glypiated) proteins to re-integrate into the plasma membrane of any target cell ( [24][25][26][27][28][29], reviewed in [30]). Based on this biochemical property it is possible to express exogenous proteins at the cell surface without the need for transfection.…”