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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.

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b. DAF and CD1c. DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c. DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b. DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c. DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b. DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.

The feasibility of using protein transfer as a means for enhancing the immunogenicity of murine tumor cells was evaluated. Glycosyl-phosphatidylinositol (GPI)-modified variants of the murine costimulators B7-1 (CD80) and B7-2 (CD86), designated B7-1.GPI and B7-2.GPI, respectively, were immunoaffinity-purified from CHO-K1 cells transfected with glutamine synthetase amplification/expression constructs encoding each of these chimeric proteins. The proteins, once purified in detergent-depleted pseudomicelles, were exogenously incorporated into the membranes of several different murine tumor lines (EL-4, SMUCC-1, BW5147.3, P815, Ag104A, and EMT6). Successful membrane painting with the B7.GPI proteins was documented by immunofluorescence and flow cytometry, and membrane integration was verified by demonstrating that the reincorporated proteins were phosphatidylinositol-phospholipase C-sensitive, glycosyl-phosphatidylinositol-phospholipase D-resistant, and refractory to removal with dimyristylphosphatidylcholine vesicles. Significantly, B7-1.GPI and B7-2.GPI could be together copainted onto EL-4 cell surfaces with no interference observed between the two. A standard in vitro proliferation assay was used to show that both of the B7.GPI proteins retained costimulator function after membrane reincorporation. These findings further validate the therapeutic potential of protein-transferred costimulator.GPIs and pave the way for their combinatorial use in animal tumor models.

Glycosyl phosphatidylinositol (GPI) lipids function as anchors of membrane proteins, and free GPI units serve as intermediates along the path of GPI-anchor biosynthesis. By using in vivo cell surface biotinylation, we show that free GPIs: 1) can exit the rough endoplasmic reticulum and are present on the surface of a murine EL-4 T-lymphoma and a human carcinoma cell (HeLa), 2) arrive at the cell surface in a time and temperature-dependent fashion, and 3) are built on a base-labile glycerol backbone, unlike GPI anchors of surface proteins of the same cells. The free GPIs described in this study may serve as a source of hormone-sensitive phosphoinositol glycans. The absence of free GPIs from the cell surface may also account for the growth advantage of blood cells in paroxysmal nocturnal hemoglobinuria.

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.

Glycosylphosphatidylinositol (GPI)-modified variants of murine B7-1 and B7-2 cell surface costimulators were produced via chimerization with alternative GPI-modification signal sequences from decay-accelerating factor (DAF). GPI anchorage was verified by demonstrating phosphatidylinositol-specific phospholipase C (PI-PLC) sensitivity of the chimeric polypeptides in both immunofluorescence/flow-cytometric and immunoprecipitation analyses. The various GPI-modified chimeric B7-1:DAF and B7-2:DAF polypeptides were shown to retain costimulator function, in both an in vitro proliferation assay and an in vivo triggering of cytotoxicity assay. The findings indicate that costimulator function for both B7-1 and B7-2 is not dependent upon native hydrophobic transmembrane anchorage. Moreover, the functionality of the GPI-modified variants in enhancing the immunogenicity of the murine T lymphoma line EL-4 suggests a novel route for generating APC-centered immunotherapeutics, including cellular cancer vaccines, that is based upon protein transfer of GPI-modified costimulators.

Alloantigen presentation by GPI-reanchored variants of the human class I MHC molecule HLA-A2.1 was studied in human cellular systems. To this end, we generated chimeric coding sequences for two GPI-modified HLA-A2.1 heavy chain derivatives. In these chimeras, the coding sequence for the HLA-A2.1 heavy chain was fused in-frame to alternative overlapping sequences from the 3'-end of human DAF containing the GPI-modification signal sequence. The encoded polypeptides HLA-A2.1:DAF-S and HLA-A2.1:DAF-L differed by 53 amino acids of additional DAF sequence in the latter. Both were detected on stably transfected C1R cell surfaces by HLA-A2.1-specific mAb, and their GPI-modification was confirmed by PI-PLC enzymatic cleavage. Immunoprecipitation analysis of surface-biotinylated C1R transfectants revealed heterodimeric association for both HLA-A2.1:DAF-L and HLA-A2.1:DAF-S heavy chains with beta 2m. Alloantigenic stimulation by, and cytotoxic recognition of, both HLA-A2.1:DAF-S/C1R and HLA-A2.1/CIR cells was observed; however, HLA-A2.1:DAF-L/C1R cells could not serve as allostimulators or allotargets. These findings establish that polymorphic human class I MHC molecules can function, when artificially GPI-reanchored, as alloantigenic targets. Moreover, the data suggest that the sequence bridging the HLA-A2 extracellular domain and the membrane can influence alloantigenic presentation.

A novel strategy for altering the adhesive properties of cells has been developed which is based upon the use of artificial adhesins. Specifically, a glycosylphosphatidylinositol (GPI)-modified variant of the cytokine macrophage colony stimulating factor (M-CSF), designated M-CSF.GPI, was expressed on the surface of human bone marrow stromal cells. A chimeric M-CSF:decay-accelerating factor expression construct was used for M-CSF.GPI expression. Cell:cell binding assays established that this artificially membrane-tethered cytokine functions as a potent cellular adhesin, allowing for enhanced binding to M-CSF receptor-expressing cellular transfectants. Antibody blocking analyses confirmed the M-CSF:M-CSF receptor dependence of the enhanced intercellular binding. This capacity to direct the cellular interactive repertoire of selected cells can in principle be applied to other cell types and other molecular pairs to be used in cell-based therapies.

A single metabolic path leading to synthesis of ether lipids is known in animal cells, the major products of which are plasmalogens. To learn whether this peroxisomal path is also responsible for the synthesis of base-resistant lipid components of glycosylphosphoinositol (GPI)-anchored membrane proteins, we have investigated the structure of anchor precursor mannolipids both in wild-type cells (CHO-K1 and a macrophage-like line, RAW 264.7) and in two corresponding mutant cells in which ether lipid biosynthesis is severely impaired. We observe that the precursor mannolipids of both the wild-type and mutant cells do not include alkylglycerol. Nevertheless, both wild-type and mutant cells express cell surface GPI-anchored placental alkaline phosphatase (AP) which includes alkali-resistant hydrophobic chains in its anchor moiety. Thus, (i) in normal AP GPI anchor synthesis, any ether-linked substituents must be added either immediately before, during, or after anchor addition to AP, and (ii) the classical peroxisomal path for ether lipid synthesis appears not to contribute to the synthesis of GPI anchors.

Mutants of bovine herpesvirus 1 that express a truncated envelope glycoprotein gIII or a gIII-human decay-accelerating factor (hDAF) chimeric protein (gIII.hDAF) were employed to evaluate the function of the transmembrane and cytoplasmic domains of the gIII molecule. Truncated gIII (i.e., lacking the transmembrane and cytoplasmic region) was readily released from infected cells and was not detected on mature virus particles. In contrast, replacement of the transmembrane and cytoplasmic domains with the carboxyl-terminal portion of hDAF restored the expression of gIII on the membranes of infected cells as well as on virion surfaces. The presence of the gIII.hDAF chimera on virus particles was also associated with normal gIII function, i.e., the mediation of virus attachment and penetration. The gIII-hDAF chimera, which is present on both infected cell surfaces and virions, could be cleaved by a phosphatidylinositol-specific phospholipase C, indicating that it was anchored in the membrane via glycosyl phosphatidylinositol. Our results from this study suggest that the transmembrane and cytoplasmic regions of the gIII molecule serve as a general membrane anchor, but they do not contain structural signals required for the specific assembly of envelope proteins into mature virions.