Electropermeabilization mediates a stable insertion of glycophorin A with Chinese hamster ovary cell membranes

Abstract

Electropulsation allowed us to incorporate glycophorin A, an integral membrane protein, into mammalian nucleated cell membranes (Chinese hamster ovary cells). The induction of stable protein association is effective only when the field intensity is higher than its threshold value, creating membrane permeabilization to small molecules. Under controlled conditions, cell viability was only slightly altered by this treatment. Pulse number and duration controlled both the number of modified cells and incorporated molecules. The phenomena was temperature dependent. An average of 5 X104 moleculeskell was bound. About 80% of cells in the pulsed population were observed to incorporate glycophorin. The protein incorporation was shown to be stable 48 h after electroassociation. Electrically bound proteins were shared between the cells after each division. As enhanced binding is detected if glycophorin is added after the pulses, it is the long-lived alteration of the membrane mediated by the pulses which supports the association.It is commonly accepted that the driving force for the spontaneous incorporation of proteins is hydrophobic effect. However, a number of other effects may support or counteract this hydrophobic effect. Generally, the free energy of protein incorporation includes four contributions which arise from a change of water structure, protein state, lipid state, bonds between protein and water or lipid molecules. Nevertheless, the central questions of how hydrophobic sequences would be transferred from an aqueous to an hydrophobic environment and how the polar regions of protein that span the membrane would be transferred across the hydrocarbon core of the bilayer, have not been emphasized. So, this makes it exceedingly unlikely that insertion of integral protein occurs directly across the lipid bilayer (Singer, 1990;Singer and Yaffe, 1990; Singer et al., 1987a, b). The membrane contribution, which has been neglected until now, necessarily appears important allowing the penetration of macromolecules with strongly polar segments. Thermodynamics of protein insertion in lipid systems imply the occurrence of the lipid matrix perturbation. Thus, protein incorporation thermodynamics ( J i m and Zakim, 1987;Jahnig, 1983) show a favourable contribution of electrostatic forces and hydrophobic forces which occur on the membrane-spanning sequence, whereas hydration forces (Pargesian and Rau, 1984) and hydrophobic forces which occur on the protein polar segments prevent incorporation. Spontaneous insertion takes place if the first component is stronger than the second one. Such a case would occur only if the membrane is in an 'excited' state.Electropulsation can create such an 'excited' state of the membrane. Recent studies reported the so-called 'electroinCorrespondence to J. Teissie,

Electroinsertion of Glycophorin A in Interdigitation-Fusion Giant Unilamellar Lipid Vesicles

“…No spontaneous insertion of glycophorin was observed without the electrical treatment. The insertion took place only in the "electropermeabilized" part of the cell surface (27,28). This observation means that the structural modification associated with electropermeabilization has brought the membrane organization to a new state in which spontaneous insertion occurs.…”

“…We showed previously that the number of electroinserted glycophorins was dependent on the bulk concentration of the protein (28). The asymmetric insertion of the protein in membranes may be due to an electrophoretic accumulation of the protein at the surface of the bilayer during the electropulsation process.…”

“…Studies have reported the so-called "electroinsertion" of proteins, having a membrane spanning sequence, such as glycophorin and CD4, into eukaryotic cell membranes by exposing the cell suspension to electric field pulses (23)(24)(25)(26)(27)(28)(29). No spontaneous insertion of glycophorin was observed without the electrical treatment.…”

“…This phospholipid has a phase T m of around 41°C. Glycophorin, which is known to be competent for electroinsertion in mammalian cell membranes and MLVs (8,(27)(28)(29), was used as a membrane protein model. Its primary structure shows a large glycosylated external domain, then a stretch of hydrophobic amino acids with a transmembraneous helical conformation and a cytoplasmic fragment.…”

Glycophorin A Protects K562 Cells from Natural Killer Cell Attack

“…This was achieved by electroinsertion, which has recently been used to insert proteins with a membrane spanning sequence into mouse red blood cell membranes (22) and into nuclear cells (23,24). We demonstrate that such modified cells are resistant to NK cell-mediated lysis.…”