title: Purification and optimization of functional reconstitution on the surface of leukemic cell lines of GPI-anchored Fc gamma receptor III.
abstract: Purified glycosyl phosphatidyl inositol (GPI)-anchored cell surface proteins can be reincorporated spontaneously into the cell membrane by incubating the cells with these proteins. This unique property provides a novel way of introducing cell surface receptors on live cell membranes without the use of gene transfection. Since any classical transmembrane cell surface protein can be converted to a GPI anchored protein by recombinant techniques, this method provides a means of studying ectodomain associated receptor functions on various cell types. Moreover, in some circumstances, it can be used to correct deficient cellular functions resulting from lack of cell surface protein expression. Using GPI-anchored Fc gamma receptor III (CD16B), a low affinity Fc gamma receptor, we have systematically studied the optimal conditions for reconstitution of a functional receptor on nucleated cells. CD16B is purified to homogeneity from neutrophil lysates by single step immunoaffinity chromatography. The purified CD16B is functionally active as evidenced by its ability to bind IgG opsonized erythrocytes. CD16B incorporation on nucleated cells is temperature dependent with an optimum of 37 degrees C. The level of expression of incorporated CD16B is also depend on the concentration of CD16B available and the duration of incubation. The incorporated CD16B retains its ability to bind ligand and also mediates endocytosis of the bound ligand. In summary, our results demonstrate that purified, functionally active GPI-anchored receptors can be expressed on desired cells in a controlled manner and retain some functional properties.
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: S
authors.initials: S
authors.lastname: Nagarajan
authors.forename: M
authors.initials: M
authors.lastname: Anderson
authors.forename: S N
authors.initials: SN
authors.lastname: Ahmed
authors.forename: K W
authors.initials: KW
authors.lastname: Sell
authors.forename: P
authors.initials: P
authors.lastname: Selvaraj
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004705:Endocytosis; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques; D007938:Leukemia / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009504:Neutrophils / Q000276:immunology*; D017452:Receptors, IgG / Q000276:immunology / Q000302:isolation & purification*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
chemical_list: D017261:Glycosylphosphatidylinositols; D017452:Receptors, IgG