Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase.

A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

Phase behavior and arrangement of molecular species in mixtures of a mixed chain and a symmetric phosphatidylethanolamine in the gel and fluid phases.

1-Octadecanoyl, 2-decanoylphosphatidylethanolamine (C(18:0)C(10:0)PE) has been reported to exhibit mixed interdigitated gel-phase packing of the phospholipid acyl chains (Mason, J.T. and Stephenson, F.A. (1990) Biochemistry 29, 590-598). In contrast, ditetradecanoylphosphatidylethanolamine (C(14:0)C(14:0)PE) packs without significant interdigitation of the phospholipid acyl chains across the bilayer center. In this report, the gel-fluid transition temperatures of C(18:0)C(10:0)PE and C(14:0)C(14:0)PE in multilamellar dispersions were determined by fluorescence anisotropy of cis-parinaric acid and trans-parinaric acid with a descending temperature scan rate of 0.67 degrees C/min. The transition mid-points detected for C(18:0)C(10:0)PE with cis-parinaric acid were 19 degrees C in water, 18 degrees C at pH 8.1, and 14 degrees C at pH 10. The phase diagram for C(14:0)C(14:0)PE and C(18:0)C(10:0)PE at pH 10 suggests complete mixing in the fluid phase and considerable immiscibility in the gel phase. Cross-linking of equimolar mixtures of C(14:0)C(14:0)PE and C(18:0)C(10:0)PE with dimethylsuberimidate at pH 10 revealed a random arrangement of the two species in the fluid phase, confirming the notion that C(18:0)C(10:0)PE and C(14:0)C(14:0)PE are miscible in the fluid phase, as determined from the phase diagram. In contrast, cross-linking of the equimolar mixture of C(18:0)C(10:0)PE and C(14:0)C(14:0)PE in the gel phase at 0 degrees C revealed a non-random arrangement, demonstrating and confirming immiscibility in the gel phase.

Detergent solubilization of the EGF receptor from A431 cells.

Functional reconstitution of purified preparations of human epidermal growth factor receptor (EGFR) requires dissociation of the protein from its plasma membrane lipid environment. Solubilization of membrane proteins in this manner requires the use of detergents, which are known to disrupt plasma membrane lipid/protein interactions. We have investigated the ability of three nonionic detergents to solubilize the human EGFR selectively, and have also analyzed the effect of these various treatments on the intrinsic tyrosyl kinase activity of the receptor. The nonionic detergent known as n-octyl glucoside (n-octyl beta-D-glucopyranoside) was found to give the best combination of selectivity, yield, and maintenance of enzymatic activity of the human EGFR.

The effect of a low essential fatty acid diet on hibernation in marmots.

We investigated the effect of an essential fatty acid (EFA)-deficient diet on hibernation patterns in yellow-bellied marmots (Marmota flaviventris). Fatty acid (FA) analysis of white adipose tissue (WAT) from animals maintained for 2 mo on the EFA-deficient diet suggested that little or no EFAs were present in the gonadal or omental fat depots. Hibernation about lengths of the EFA-deficient animals were significantly shorter (P < 0.01) than control animals. Stated another way, these animals aroused twice as frequently compared with control animals and used more energy to survive winter. Analysis of WAT composition and blood samples revealed that animals were highly lipolytic during winter. Furthermore, the release of FAs was not random: linoleate (cis-9,cis-12-octadecadienoic acid; 18:2, a diene EFA) was significantly (P < 0.05) under-represented in venous outflow from the gonadal WAT pad based on the percentage of this species in WAT. The concentration of saturated FAs was higher than that predicted from the WAT-FA composition. We conclude that linoleate is preferentially retained within WAT and that concentrations of this EFA may influence hibernation behavior. Thus EFAs may have a thermoregulatory role in hibernation in addition to their role as essential precursors for physiologically important lipids after hibernation is over.

Is receptor oligomerization causally linked to activation of the EGF receptor kinase?

Transduction of a signal from an extracellular peptide hormone to produce an intracellular response is often mediated by a cell surface receptor, which is usually a glycoprotein. The secondary intracellular signal(s) generated after hormone binding to the receptor have been intensively studied. The nature of the primary signal generated by ligand binding to the receptor is understood less well in most cases. The particular case of the epidermal growth factor (EGF) receptor is analyzed, and evidence for or against two dissimilar models of primary signal transduction is reviewed. Evidence for the most widely accepted current model is found to be unconvincing. Evidence for the other model is substantial but indirect; a direct test of this model remains to be done.

Effects of gangliosides GM3 and De-N-acetyl GM3 on epidermal growth factor receptor kinase activity and cell growth.

Previously it was reported (Bremer, E.G., Schlessinger, J., and Hakomori, S.-I. (1986) J. Biol. Chem. 261, 2434-2440) that ganglioside GM3 inhibited epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor in Triton X-100-treated preparations of human epidermoid carcinoma (A431) cell membranes. In addition, these authors reported that GM3 inhibited the growth of A431 cells. In contrast, a modified ganglioside, de-N-acetyl GM3, enhanced the EGF-dependent tyrosine kinase activity of the EGF receptor. In this work and in subsequent studies (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S.-I. (1988) J. Biol. Chem. 263, 6296-6301), the tyrosine kinase activity of the receptor from A431 cell membranes was assayed in the presence of Triton X-100. In this report, we confirm that GM3 inhibited and de-N-acetyl GM3 stimulated EGF receptor autophosphorylation in the presence of Triton X-100. However, in the absence of detergents, ganglioside GM3 inhibited EGF-stimulated receptor autophosphorylation, whereas de-N-acetyl GM3 had no effect on EGF-stimulated receptor autophosphorylation. The effects of these gangliosides on receptor autophosphorylation were measured in both A431 cell plasma membranes and in 3T3 cell membranes permeabilized to [32P]ATP by a freeze-thaw procedure, in intact A431 cells permeabilized with alamethicin, and in intact A431 cells grown in the presence of [32P]orthophosphate. Thus, the inhibitory effect of GM3 on receptor autophosphorylation was demonstrated in the presence and in the absence of detergent; the stimulatory effect of de-N-acetyl GM3 was observed only in the presence of detergent. We also demonstrate that ganglioside GM3 inhibited EGF-stimulated growth of transfected murine fibroblasts (3T3) that express the gene for human EGF receptor (Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan, I., and Lowy, D. R. (1989) J. Cell. Biochem. 39, 153-166). De-N-acetyl ganglioside GM3 had no effect on the growth of these cells. Growth of control fibroblasts, which lack endogenous EGF receptors (Pruss, R. M., and Herschman, H. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921), was not affected by the presence of either ganglioside. Similarly, ganglioside GM3, but not de-N-acetyl ganglioside GM3, inhibited the EGF-dependent incorporation of [3H]thymidine into DNA by transfected fibroblasts. Incorporation of labeled thymidine into DNA of control fibroblasts was not affected by the presence of either ganglioside. These studies indicate that ganglioside GM3, but not its deacetylated analogue, can affect EGF receptor kinase activity in intact membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma and white adipose tissue lipid composition in marmots.

White adipose tissue biopsies and plasma samples were obtained from hibernating yellow-bellied marmots (Marmota flaviventris) maintained in the laboratory. In addition, biopsies and plasma samples were obtained from normothermic animals in the field and laboratory. Measurement of plasma free fatty acid (FA) levels indicated that winter laboratory animals exhibited increased lipolysis. Additionally, analysis of white adipose tissue triacylglycerol revealed that the FA composition of the storage fat in animals maintained on the standard laboratory diet is remarkably simple and uniform between different adipose depots in the same animal. Three FAs (palmitic, oleic, and linoleic acids) made up greater than 95% of the total. Triene (alpha-linolenate) was found in newly captured animals, but the percentage of this FA decreased rapidly when the animals were maintained on the standard laboratory diet. Throughout the hibernation season (October to April), white adipose tissue-saturated FA percentage decreased, monoene percentage remained constant, and diene percentage increased. Analysis of plasma FA composition suggested that these animals tended to metabolize saturated FAs from stored lipid during hibernation and that dienes were mobilized briefly after the last arousal from hibernation in spring. From these observations, we hypothesize that marmots preferentially metabolize saturated fats during the hibernation period and that essential FAs of the omega 6 series tend to be metabolized more slowly than other FAs. These characteristics suggest that marmots are a valuable animal model in which to study lipid metabolism.

A fluorescence-quenching assay for measuring permeability of reconstituted lens MIP26.

A sensitive fluorometric assay for measuring permeability of liposomes containing bovine lens MIP26 has been developed and characterized. Lens membrane proteins were isolated and incorporated into artificial membranes composed of bovine brain phospholipids, sphingomyelin and the fluorescent probe, N-4-nitrobenzo-2-oxa-1,3 diazole phosphatidylethanolamine (NBD-PE). Quenching of the probe with cobalt chloride allowed the measurement of liposome permeability in the presence and absence of lens membrane proteins. Liposomes containing the putative gap junctional polypeptide known as Major Intrinsic Protein 26 (MIP26) were shown to be more permeable than those not containing the MIP26 protein. Permeability was shown to be positively correlated with the amount of MIP26 in the liposomes and with increasing purity of the protein. This method offers a sensitive assay for channel function of the putative junction protein, and provides further evidence that MIP26 from lens membranes is a gap junctional polypeptide.

Synthesis and characterization of N-parinaroyl ganglioside GM1: effect of choleragen binding on fluorescence anisotropy in model membranes.

N-cis-Parinaroyl ganglioside GM1 and N-trans-parinaroyl ganglioside GM1 were synthesized and characterized by HPLC, TLC, component analysis, absorbance spectroscopy, and proton NMR spectroscopy. Steady-state fluorescence anisotropy of the purified compounds, incorporated into phosphatidylcholine liposomes, was measured in the presence and absence of choleragen (cholera toxin) and choleragenoid (cholera toxin B subunit). In gel-phase liposomes, anisotropy measurements indicated that the motion of the parinaroyl ganglioside was not affected by addition of choleragen or choleragenoid. In fluid-phase liposomes, however, addition of toxin resulted in increased anisotropy (decreased rotational motion) of the fluorescent gangliosides. This decreased motion was not observed with other parinaroyl lipid probes, such as phosphatidylcholine, glucosylceramide, or free fatty acids, indicating that the effect was due to specific ganglioside/toxin interactions. Varying the amount of ganglioside or the amount of toxin suggested that the effect of toxin on probe motion was saturable at approximately 1 choleragen (or choleragenoid) molecule/5 ganglioside molecules. These results are consistent with previous hypotheses regarding the ganglioside/choleragen interaction and indicate that parinaroyl ganglioside probes will be useful in elucidation of the molecular details of this interaction.

Thermotropic behavior of mixtures of glycosphingolipids and phosphatidylcholine: effect of monovalent cations on sulfatide and galactosylceramide.

The thermotropic behavior of both sulfatide (3-sulfogalactosylceramide) and galactosylceramide in dielaidoylphosphatidylcholine (DEPC) liposomes was studied, using steady-state fluorescence polarization of parinaric acid isomers. The glycosphingolipid (GSL) concentration of the liposomes was varied from 0 to 100%, and phase diagrams were constructed. The data indicate that sulfatide and DEPC are immiscible in the gel phase at sulfatide mole ratios of less than 0.30. The temperature of onset of the gel-liquid-crystalline phase transition is higher in K+ -containing buffer than in osmotically equal Na+ -containing buffer. Similar measurements, using galactosylceramide, a neutral GSL, indicated that this lipid and DEPC are immiscible in the gel phase at galactosylceramide mole ratios of less than 0.40. In contrast to the results obtained with sulfatide, onset temperatures are identical in Na+- or K+-containing buffers. The phase properties of sulfatide/DEPC mixtures are shown to depend on the cation only when the sulfatides contain hydroxy fatty acids. Our observations indicate that physiologically relevant concentrations of monovalent cations affect motion and distribution of sulfatide in biological membranes and further implicate this GSL as an important determinant of function of the Na+,K+-ATPase. A preliminary report of these data [Rintoul, D.A., Welti, R., & Song, W. (1988) Biophys. J. 53, 126a].

Early events in polyomavirus infection: fusion of monopinocytotic vesicles containing virions with mouse kidney cell nuclei.

The entry of polyomavirus enclosed in monopinocytotic vesicles into mouse kidney cell nuclei was studied and evidence for a fusion mechanism was obtained. In vivo studies using the fluorescent lipophilic dye diI-C16(3) as a plasma membrane label showed that polyomavirus-infected nuclei accumulate plasma membrane, while uninfected or polyoma capsid-infected nuclei do not. Further evidence for fusion was obtained with electron microscopy of thin sections of infected mouse kidney cells. These specimens showed accumulation of plasma membrane in the outer nuclear membrane as well as evidence of recent fusion events. The polyoma virions (capsid proteins) were seen to accumulate on the inner nuclear membrane and in the nucleus and were identified by immunogold staining of the thin sections. The combined results of the in vivo dye studies and thin section immunoelectron microscopy studies provide evidence for a fusion mechanism for polyomavirus entry into mouse kidney cell nuclei.

Physical properties of membranes and membrane lipids from the fiber cell of the U18666A-cataractous rat.

Steady state fluorescence anisotropy of parinaric acid probes was used to examine lipid motion in membranes and membrane lipids from U18666A-induced cataractous rat lenses. Cortical and nuclear fractions were examined separately. The drug treatment resulted in an increase in the sterol/phospholipid ratio in the nucleus and a decrease in this ratio in the cortex. Fluorescence anisotropy of trans parinaric acid (tPnA) suggested that membranes from the cortex of cataractous lenses were more ordered than the cortical membranes from control or treated-but-clear lenses. Membranes from the nucleus of control lenses were more ordered than membranes from the cortex of controls. Nuclear membranes from cataractous lenses were slightly less ordered than nuclear membrane from control or treated-but-clear lenses. Similar experiments using liposomes prepared with membrane lipid from lens cortex showed that membrane lipids from treated lenses, control lenses, and treated-but-clear lenses had similar fluorescence anisotropy profiles, suggesting that cortical lipids had similar order. Conversely, fluorescence anisotropy of parinaric acid probes in liposomes prepared from nuclear fractions showed a slight increase in lipid order from control to treated-but-clear to cataractous preparations. These results are interpreted to indicate the presence of an alteration in lipid-protein interactions in cortical membranes in the cataractous lenses; this results in more ordered membranes at the physiological temperature in these lenses. The increase in the order of cortical membranes from cataractous lenses is dependent on the presence of membrane proteins rather than lipids, since it is not seen in the liposome preparations (which are protein-free).(ABSTRACT TRUNCATED AT 250 WORDS)

N-parinaroyl glycosphingolipids: synthesis and characterization of novel fluorescent probes of membrane structure.

N-Parinaroylceramides and -glucocerebrosides were synthesized and characterized. These fluorescent glycolipids were found to be nonperturbing membrane lipid probes, which partitioned preferentially into fluid-phase phosphatidylcholine (PC) in liposomes containing both fluid and solid-phase PC. N-Parinaroylglucocerebroside, parinaroyl-PC, and free parinaric acid were used to analyze the motion and distribution of glucocerebroside and ganglioside GM1 in liposomes composed of these glycosphingolipids (GSL) and 1-stearoyl-2-oleoyl-PC (SOPC). Steady-state fluorescence anisotropy of these probes indicated that the neutral glucocerebroside formed solid-phase domains in SOPC liposomes; these domains contained little or no PC. In contrast, the negatively charged ganglioside GM1 was miscible with fluid-phase PC. Incorporation of GM1 into SOPC liposomes resulted in an increase in the transition temperature of the mixture; no transition was observed in either of the pure GSL used over the temperature range from 5 to 70 degrees C. These data indicate that the glucocerebroside probes may be specific for sphingolipid domains in mixed PC/GSL membranes.

The role of gangliosides in the interaction of a growth inhibitor with mouse LM cells.

We have isolated and characterized glycopeptides, derived from mouse and bovine cerebral cortex cells, that inhibit protein synthesis and cell growth of normal but not transformed cells. The inhibitor binds to target cell surfaces, and gangliosides have previously been shown to influence cell sensitivity to the glycopeptides. Preincubation with 3.0 micrograms/ml ganglioside GM1 at 0 degrees C for 3 hr sensitized the mouse L-cell line to the inhibitor, as determined by protein synthesis assays. Preincubation of LM cells with ganglioside GM1 alone did not affect protein synthesis rates. In addition, the gangliosides GD1a and GM3 also sensitized the LM cells to the protein synthesis inhibitory effect of the glycopeptide inhibitor. Binding experiments were performed with 3T3 (sensitive) and LM (insensitive) cells to determine if sensitivity to the glycopeptide inhibitor was reflected in binding of the inhibitor to these cells. Binding of 125I-labeled inhibitor to 3T3 cells was maximal after 60 min at 0 degrees C and saturable at approximately 1 X 10(4) molecules/cell. Furthermore, binding of the inhibitor was dose-dependent, with half-maximal binding at 1.5-2.0 nM and saturation at 8.0-10.0 nM. Scatchard plot analysis indicated that the Kd was about 1 X 10(-9) M and that there are 1 X 10(4) receptors/cell. Binding of the inhibitor to LM cells was maximal after 30 min at 0 degrees C and saturation occurred at 5 X 10(3) molecules/cell. We then examined the possibility that gangliosides are the cellular receptor or co-receptor for the glycopeptide inhibitor. Binding of the inhibitor to ganglioside GM1 was first examined after the ganglioside had been preadsorbed to polystyrene tubes. These experiments indicated that the ganglioside did not bind the inhibitor. Ganglioside-containing liposomes from phosphatidylcholine or LM cell membrane components were also prepared; these artificial membranes did not bind appreciable amounts of the iodinated inhibitor. Competition experiments showed that the gangliosides GM1 and GD1a did not neutralize the protein synthesis inhibitory activity of the glycopeptides, indicating that gangliosides do not directly interact with the glycopeptide inhibitor. In addition, binding of the inhibitor to LM cells preincubated with ganglioside GM1 was studied. Although the binding of the inhibitor to LM cells was one-half that observed for 3T3 cells, incorporation of exogenous gangliosides into LM cells did not result in increased binding of the inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

Reconstitution of MIP26 from single human lenses into artificial membranes. I. Differences in pH sensitivity of cataractous vs. normal human lens fiber cell proteins.

Reconstitution of the lens fiber cell protein known as MIP26 into liposomes composed of heterologous phospholipids was achieved; this protein renders the liposomes permeable to low molecular weight compounds. MIP26 from either bovine or human lenses was capable of forming channels in artificial membranes. The assay technique was sufficiently sensitive to allow reconstitution of MIP26 from single human lenses, enabling us to examine the function of channels from either cataractous or age-matched normal lenses. Decreases in pH can cause these channels to close, analogous to the hypothesized channel closing in the in vivo situation. The pH optimum of reconstituted channels in liposomes containing MIP26 from bovine lenses or normal human lenses is very sharp; but is substantially broadened if the liposomes contain MIP26 from cataractous human lenses. This latter result suggests a functional alteration in human lens membranes which is correlated with the development of human senile cataract.

Involvement of plasma membrane lipid structural order in adriamycin resistance in Chinese hamster lung cells.

Plasma membrane preparations from Chinese hamster lung cells, which are resistant to the antitumor agent Adriamycin, were analyzed using fluorescence polarization of the membrane lipid probe trans-parinaric acid. The results of these studies reveal that membranes from several drug-resistant isolates have a substantial decrease in lipid structural order relative to membranes from drug-sensitive cells. Additional studies have shown that certain isolates are unstable and undergo a sequential phenotypic reversion after continuous passage in culture. Thus, we have identified cells which have reverted for membrane lipid physical changes but which still remain highly resistant to Adriamycin. At later passages, these cells are found to revert to drug sensitivity. These results indicate that an alteration of plasma membrane lipid structural order is not an essential component of the Adriamycin-resistant phenotype. However, in certain isolates, drug resistance and changes in membrane physical properties are both associated with an unstable genetic element.

Changes in chick corneal lipids during development.

Chick corneas from days 9 through 18 of embryonic development were analyzed for phospholipid and glycolipid content, and for phospholipid and ganglioside classes using chromatographic techniques. Little change in phospholipid headgroup classes was detected during this time span, with the exception of a steady increase in the content of phosphatidylserine from 12 to 18% of the total. Changes in glycolipid content also were observed, with a maximum in the sphingolipid/phospholipid ratio at day 14. This increase was correlated with the appearance of highly sialylated gangliosides at days 14 and 15; these ganglioside species then declined in abundance until they were no longer detectable at day 18. These results indicate that gangliosides with high sialic acid content undergo stage-specific developmental cycles in the chick cornea. Furthermore, the data suggest that phosphatidylserine content may be a sensitive biochemical measure of corneal innervation.

Luteolysis-induced changes in phase composition and fluidity of bovine luteal cell membranes.

X-ray diffraction, fluorescence polarization of trans-parinaric acid, and fluorescence photobleaching recovery of dioctadecyltrimethyneindolecarbocyanine have been used to characterize the phase composition and liquid phase fluidity of bovine luteal cell membranes and membrane lipids for functional corpora lutea collected at midcycle and for regressing corpora lutea collected after treatment with prostaglandin F2 alpha. These results support previous observations of gel phases in microsomal preparations of regressed luteal cells at physiological temperatures and further suggest that the plasma membrane may be the main source of this gel phase. Analysis of the overall lipid composition of the microsomal preparations from these cells indicates a role for sphingomyelin, in the presence of cholesterol, for the generation of a gel phase at physiological temperatures.

Isolation and characterization of filipin-resistant LM cell variants not auxotrophic for sterol.

A series of LM cell variants resistant to filipin, but not auxotrophic for sterol, was isolated by plating mutagenized, filipin-treated cells on soft agar medium containing no sterol. Cloned variants were assayed for growth in the presence and absence of sterol or unsaturated fatty acid. Filipin-resistant clones whose growth rate was unaffected by the addition of sterol to the medium were further analyzed. Variants were cultured in minimal medium, and plasma membranes prepared from these cultures were analyzed for sterol content, phospholipid head group composition, and unsaturated fatty acyl content. All variants examined showed a decrease in membrane sterol in conjunction with an increase in unsaturated fatty acyl chains in the membrane phospholipids. In addition, several variants exhibited alterations in phospholipid head group composition, including changes in the phosphatidylcholine/phosphatidylethanolamine ratio, or a decrease in sphingomyelin content. These observations imply that several different metabolic lesions can give rise to decreased plasma membrane sterol content (and hence to filipin resistance). The range of phospholipid alterations observed in these sterol prototrophs emphasizes the complex interrelationship between membrane sterol and phospholipid structure.