Bernadine J. Wisnieski

Alterations of characteristic temperatures for lectin interactions in LM cells with altered lipid composition

Abstract Alteration of the fatty acid composition of mouse LM cell lipids dramatically affected the concanavalin A binding and concanavalin A-mediated hemadsorption properties of these cells. A critical temperature for these two concanavalin A related phenomena observed at 15–19° in cells with unaltered fatty acid composition was shifted to 22–28° for cells containing a higher proportion of saturated fatty acids and lowered to 7–11° for cells containing polyunsaturated fatty acids substituted for monoenoic unsaturated fatty acids. In contrast, a second critical temperature (at 5–7°) observed for concanavalin A binding and concanavalin A-mediated hemadsorption to LM cells was essentially unchanged by alterations in cellular lipid fatty acid composition. We conclude that a change in membrane lipid freezing point is responsible for the higher critical temperature (15–19°), and factors other than lipid melting properties, perhaps cytoskeleton structure, contribute to the lower critical temperature (5–7°) for lectin interactions with the exposed surface of LM cells.

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of Sendai virus as fusogen.

A method of introducing enzymes into the cytoplasm of fibroblasts in culture is described. Erythrocytes obtained from normal and arginase-deficient individuals were loaded with arginase in vitro and fused to arginase-deficient mouse and human fibroblasts. Erythrocyte ghost-fibroblast fusion was quantified by a 14C-radioactive assay for arginase in solubilized fibroblasts. Fusion was successfully induced by Sendai virus and also by the isolated glycoproteins of Sendai virus. After fusion the arginase activity associated with the Fibroblasts was 700--1500 U of arginase/mg of cell protein; this enzyme activity was 5- to 10-times higher than that normally found in the fibroblasts. The enrichment in arginase activity indicated that between four and ten ghosts had fused per fibroblast. The use of isolated viral proteins to mediate the transfer of enzymes into cells in vivo might alleviate clinical complications inherent in the use of whole virions. The enzyme replacement technique described in this report for a hyperargininemic model cell system should be applicable to the group of inborn errors of metabolism characterized by deficiency of an enzyme normally localized in the cytoplasmic compartment of cells.

Labeling of the active subunit of cholera toxin from within the membrane bilayer

Abstract Using the photoreactive glycolipid probe 12-(4-azido-2-nitrophenoxy)-stearoylglucosamine-[1-14C], we have effected the radiolabeling of the active A1 subunit of cholera toxin from within the membrane bilayer. The membrane employed as a target was the envelope of Newcastle disease virus which contained the photoreactive probe. Radiolabeling of the A1 subunit occurred after cholera toxin and virus were incubated together for 15 min at 37° and then irradiated at 366 nm for 1 min. Labeling of A1 did not occur when cholera toxin was irradiated in a solution of probe without virus or when the 15 min incubation with virus was performed at 0° instead of at 37°.

Characterization of a glycoprotein fusogen isolated from sendai virus

After isolation from Sendai virus, the glycoproteins HN and F retained their ability to induce hemagglutination and both heterologous and homologous cell-cell fusion. Both methods for demonstrating cell fusion indicated that the isolated HN and F glycoproteins compared favorably with whole Sendai virus as a fusogen. Conditions affecting the degree of fusion were examined and optimized. Whole virus and isolated glycoprotein preparations were characterized by electron microscopy and by SDS-polyacrylamide gel electrophoresis. Lipid analysis of the glycoprotein preparations by thin layer chromatography and gas chromatography/mass spectrometry indicated that they were partially lipid-depleted during the isolation protocol and the ratio of cholesterol to phospholipid was higher than in the whole virus. A complete fatty acid analysis was performed on lipid extracts from whole virus and from glycoprotein preparations. Detergent was removed from the glycoproteins by dialysis and by incubation with Amberlite XAD-2 resin. The detergent content of the glycoprotein preparations was monitored by gas chromatography and with [3H]Triton X-100. Both methods showed that virtually all (greater than or equal to 99.8%) of the originally added detergent was removed. Electron microscopy of the negatively-stained HN and F preparations showed primarily spherical particles 120 +/- 20 A in diameter (range 80-250 A). Since no organization reminiscent of envelopes could be demonstrated, we conclude that the fusogenic activity of Sendai virus resides in the glycoproteins per se rather than in bilayer integrated lipid-protein complexes.

The Role of Lipids in Virus—Cell Interactions

Enveloped viruses are coated with a lipid matrix of varying complexity. This is present in a bilayer and is an essential constituent since treatment with lipid solvents, detergents or lipase inactivates infectivity and hemagglutination (Kuwert et al., 1968). Lipid composition has been closely studied in only a few viruses, examples being VSV (McSharry and Wagner, 1977), rabies virus (Blough et al., 1977; Diringer et al., 1973; Schlesinger et al., 1973) and Uukuniemi virus (an Arbovirus, Renkonen et al., 1973). An early review of lipid composition in viruses was provided by Blough and Tiffany (1973). The viral lipid composition may reflect that of host membranes, since it is often obtained during a budding process and the lipid composition of some viruses varies widely after replication in different cell systems (Klenk and Choppin, 1969, 1970; McSharry and Wagner, 1971 ; Semmel et al., 1975). The production of infectious paramyxoviruses is strongly dependent on the lipid composition of the host (Klent and Choppin, 1970b; St. Gerne et al., 1977).