Membrane proteins in human erythrocytes during cell fusion induced by oleoylglycerol

Abstract

1. The fusion of human erythrocytes into multicellular bodies that is induced by microdroplets of oleoylglycerol was investigated by optical and electron microscopy, and by gel electrophoresis of membrane proteins. 2. At the highest concentrations of oleoylglycerol and Ca(2+) used, at least 80% of the cells fused after 30min at 37 degrees C and only about 5% of the cells had completely lysed; the shapes of fused multicellular bodies were usually retained in ;ghosts' prepared by hypo-osmotic lysis. 3. The rate of cell fusion was related to the concentration of Ca(2+), although some cells fused when no exogenous Ca(2+) was present. 4. Interactions of microdroplets of oleoylglycerol with the cells led to abnormalities in the structural appearance of the erythrocyte membrane; subsequent membrane fusion occurred, at least in some instances, at the sites of the microdroplets. 5. The intramembranous particles on the P-fracture face of the treated cells were more randomly distributed, but not significantly increased in number by comparison with the control cells. 6. Gel electrophoresis of the proteins of ;ghosts' prepared from fused human erythrocytes showed a production of material of very high molecular weight, the development of a new component in the band-3 region, an increased staining of bands 4.3 and 4.5, and a new component moving slightly faster than band 6. 7. Bands 2.1-2.3 were altered, band 3 was decreased and band 4.1 was lost. 8. Most, but not all, of the changes in the membrane proteins appeared to result from the entry of Ca(2+) into the cell. 9. 1-Chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one partially inhibited both cell fusion and the associated decrease in band-3 protein. 10. The possibility that proteolytic degradation of membrane proteins may be involved in cell fusion induced by oleoylglycerol is considered, and some implications of this possibility are discussed.

Isolation and partial characterization of three acidic proteinases in erythrocyte membranes

“…Among the questions raised by such multiplicity of proteinases, a major one is that of defining specific roles for each proteinase in terms of protein substrates, accessibility of soluble proteins to their site of degradation, selective mechanisms of regulation etc. Thus, for instance, Quirk et al (1978) have reported that band 3, the major protein of erythrocyte membranes, may be a substrate for a specific proteinase activity in erythrocytes induced to fuse by treatment with oleoylglycerol. A possibly related system has been described by King & Morrison (1977), who have attributed some significant changes in membrane proteins to a Ca2+-activated proteinase.…”

Proteinases and cell fusion

“…In ghosts of human erythrocytes, the immobility of the intramembranous particles is well known and aggregation, similar to that of fig3c, is seen only after partial removal of spectrin [21]. When intact human erythrocytes were fused by oleoylglycerol, a loss of micro-aggregation in the intramembranous particles of the treated cells implied that the particles were at least partially free to move [22]. Figure 3d shows that the proteinase inhibitor PMSF, which inhibited cell fusion induced by subtilisin Carlsberg (fig2c), also inhibited the loss of intra-membranous particles and their thermally-induced, lateral movement in the membranes of enzyme-treated cells.…”

Components involved in virally mediated membrane fusion and permeability changes

“…However, it is much more likely that Ca2+ entry is a consequence, not a cause, of an altered membrane, since chemical fusogens increase membrane permeability in general, as demonstrated by Knutton & Pasternak (1979) and by Blow et al (1979) themselves. Moreover EDTA does not abolish cell-cell fusion of human erythrocytes (Quirk et al, 1978). What is probably true is that the stability of giant cells (polykaryons) requires the presence of some Ca , Table 4.…”