DE19946737A1 - Use of gangliosides to modulate sphingolipid cholesterol microdomains - Google Patents

Abstract

The use of gangliosides to prepare an agent for modulating sphingolipid cholesterol microdomains is described.

Description

The invention relates to the use of gangliosides for the preparation an agent for modulating sphingolipid cholesterol microdomains.

Sphingolipid-cholesterol microdomains, which are also called sphingolipid-cholesterol rafts, are lateral arrangements of specific lipids, in particular sphingolipids including gangliosides and cholesterol, and proteins in cell membranes (K. Simons et al., Nature, Vol. 387 ( 1997 ), 569-572; T. Friedrichson et al., Nature, Vol. 394 ( 1998 ), 802-805). They are small and highly dynamic structures that are formed by the attractive forces between sphingolipids and cholesterol. Various proteins are associated with the sphingolipid cholesterol structures, including GPI-APs (glycosyl-phosphatidyl anchor proteins), Src family kinases, influenza virus hemagglutinin (HA) and caveolin-1. Most of the proteins associated with the sphingolipid cholesterol rafts contain post-translational lipid modifications, with GPI-anchored proteins anchored in the cell membrane via a lipid unit, whereas kinases of the Src family, NO synthases (NOS), HA and caveolin are acylated . The proteins associated with the rafts are usually poorly soluble in cold, non-ionic detergents such as Triton X-100, which may be due to their lipophilic nature. Triton-insoluble complexes have been defined in terms of their function as rafts that are specifically enriched in certain lipids, especially sphingolipids including gangliosides, and cholesterol, while other lipids such as glycerophospholipids are selectively absent in the Triton extracts (Brown et al., Trends Cell Biol. 2 ( 1992 ), 338-343; Fiedler et al., Biochemistry 32 ( 1993 ), 6365-6373).

The existence of sphingolipid-cholesterol microdomains or rafts in living cells, in particular the existence of microdomains of GPI-anchored proteins in living cells, which can be influenced by the administration of cholesterol, has recently been established by a chemical cross-linking approach (Friedrichson et al. , supra) and a fluorescence energy transfer method (Varma et al., Nature 394 ( 1998 ), 798-802). It has also been postulated that rafts play an important role in membrane transport and signaling properties as platforms to which proteins can bind.

An object of the invention was therefore to provide a means for modulating Provide sphingolipid cholesterol microdomains (namely in particular their structure or / and properties) in order to be able to respond to them To be able to specifically influence microdomain-influenced processes.

This object is achieved by the use of Gangliosides for the preparation of an agent for modulating sphingolipid Cholesterol microdomains.

In the context of the present invention, it was found that with Proteins associated with sphingolipid cholesterol, in particular GPI-anchored proteins in the microdomains in the form of cross-linked Clusters are present on the surface of living cells. Through the Addition of gangliosides will cross-link with the rafts associated, in particular GPI-anchored proteins inhibited and above in addition, their solubility in detergents increases.

The administration of gangliosides thus leads to a change in the structural structure of the microdomains so that gangliosides  Modulation of sphingolipid cholesterol microdomains and those there operations taking place can be used.

It is believed that by the administration of gangliosides Proteins, especially GPI-anchored proteins, from the sphingolipid Cholesterol microdomains are ousted by the attractions between the anchor group and the surrounding lipids. As a result, the sphingolipid cholesterol microdomains proteins present when gangliosides are added from their lipid environment be detached and are no longer associated with the rafts.

As a result, the addition of exogenous gangliosides changes e.g. B. the solubility of GH-DAF (growth hormone (GH; growth hormone) linked to the GPI anchor of the DAF (Decay accelerating factor)), a glycosyl-phosphatidylinositol-anchored growth hormone form, in detergents. It is known that GPI-anchored proteins are essentially insoluble in non-ionic detergents (Brown et al., Cell 68 ( 1992 ), 533-544), while reducing the cholesterol level in the cells detergent solubility of GPI-APs (Cerneus et al., J. Biol. Chem. 268 ( 1993 ), 3150-3155; Hanada et al., J. Biol. Chem. 270 ( 1995 ), 6254-6260). This means that the interaction forces between the lipid components and the proteins of the microdomains are greater than the dissolving forces due to non-ionic detergents. However, if the microdomains are changed or destroyed by removing the cholesterol component, the interaction forces between the lipids and the proteins are reduced and the proteins can be detached from the rafts by non-ionic detergents.

Surprisingly, it has now been found that when adding Gangliosides, which are also a lipid component of the rafts, the On the contrary, the addition of gangliosides to the cells Detergent solubility of GH-DAF increased. This finding is unexpected  since one would assume that increasing the concentration of one in lipids occurring in the sphingolipid cholesterol microdomains Would stabilize these domains. In fact, the encore bothers of gangliosides, however, the cross-linking of the proteins and increases the Detergent solubility from GH-DAF. Gangliosides thus cause one Modulation of the microdomains, in particular a displacement of Proteins, especially GPI-APs from the rafts.

This is confirmed by gangliosides copatching HA and Disrupt GH-DAF. Copatching two microdomain components is one Follow the coalescence of a common lipid microdomain.

The modulation of the microdomains by the gangliosides is preferred specific and reversible, d. H. when the gangliosides are removed, again preserve the original, unchanged structure of the microdomains.

According to the invention, preference is therefore given to the use of Gangliosides the localization of proteins on sphingolipid cholesterol Affects microdomains, especially the localization of Anchor proteins, acylated proteins, Src kinases and / or cholesterol or GPI-anchored proteins. By adding gangliosides, the Arrangement of microdomains present in sphingolipid cholesterol Proteins changed, resulting in modulation and / or change in Structure and / or structure of the sphingolipid cholesterol microdomains leads. Cluster degradation of the proteins is particularly preferably effected.

The gangliosides used according to the invention are preferably selected from bovine brain gangliosides, GM ₁ , GD1a, GDib, GD3, GM2, GM3, GQ1a, GQ1b or / and globosides. Exogenous gangliosides which are already part of the sphingolipid-cholesterol microdomains to be treated are particularly preferably used.

According to the invention, the term “modulation” includes any structural and chemical change in the microdomains, especially a change in the structural structure and / or in the composition of the individual Components of the microdomains. The modulation of the sphingolipid For their part, cholesterol microdomains preferably cause a change the membrane transport, signal transmission and / or Cell adhesion properties and / or a change in enzymatic Processes. These effects are achieved through the use of Gangliosides for modulation, in particular for changing the Structure caused by sphingolipid cholesterol microdomains. So can be caused by a change in sphingolipid cholesterol rafts exogenously added ganglioside signaling properties to be influenced. In addition, it was found that the addition of Gangliosides to cells the degree of autophosphorylation of many kinases changed. The sphingolipid cholesterol microdomains thus represent Platforms for various processes, especially for Signal transmission processes, which according to the invention by modulating the Microdomains with gangliosides can be influenced.

GPI-anchored proteins associate e.g. B. with tyrosine kinases of the Src family (Brown, Trends Cell Biol. 2 ( 1992 ), 338-342). In addition, cell signaling proteins are concentrated in the caveolae, plasma membrane indentations involved in sequestering and organizing raft lipid domains. Many signaling molecules are associated with sphingolipid cholesterol rafts or caveolae, such as PDGF (platelet derived growth factor), EGF (epidermal growth factor), protein kinase C and the insulin receptor. The signal transmission pathways mediated by these molecules can be inhibited by the exogenous addition of gangliosides. It has now been found that these signal transmission paths are modulated by gangliosides, e.g. B. a cluster degradation of protein-containing microdomains, in particular of GPI-AP containing microdomains.

Further signal transmission processes depend on the arrangement of lipid and protein components in the rafts. Modulation, in particular degradation of rafts by exogenous administration of gangliosides can e.g. B. can be effected to antagonize a microdomain-dependent signal transmission. These signal transmission processes include an antibody-induced patching of GPI-anchored PLAP (placental alkaline phosphatase), which leads to an accumulation of the Src-like tyrosine kinase fyn (Harder et al., J. Cell. Biol. 141 ( 1998 ), 929 -942). Furthermore, physiological reactions induced by microdomain clusters have been described for lymphocytes (Brown, Curr. Opin. Immunol. 5 ( 1993 ), 349-354). An antibody-induced cross-linking of GPI-anchored proteins leads to the activation of T-lymphocytes (Thomas et al., J. Biol. Chem. 267 ( 1992 ), 12317-12322) and the cross-linking of the IgE receptor FcεRI causes an allergic reaction including secretion of histamine in mast cells (Holowka et al., Ann. Rev. Biophys. Biomol. Struct. 25 ( 1996 ), 73-80; Field et al., J. Biol. Chem. 272 ( 1997 ), 4276- 4280). Gangliosides can also be used to treat diseases that are associated with cell movement, such as combating tumors or metastases. Tumor cells use the above mechanism to escape the immune response. Processes mediated by rafts can thus be influenced by gangliosides, such as T-time activation in the presence of tumor cells. The use of gangliosides according to the invention makes it possible to modulate functions which are influenced by sphingolipid-cholesterol microdomains and thus to influence them in a targeted manner.

It was also found that when adding gangliosides Cells an increase in autophosphorilation by various Protein kinases can be observed.  

In addition, the gangliosides can be used as a means of elucidating the pathogenesis of diseases associated with activities on sphingolipid cholesterol microdomains. It has recently been shown that a reduction in the cholesterol of hippocampal neurons inhibits the secretion of the β-amyloid peptide, with the result that the sphingolipid cholesterol microdomains play an important role in the proteolytic processing of the amyloid precursor protein (APP) in Alzheimer's Play disease (Simons et al., Proc. Natl. Acad. Sci. USA 95 ( 1998 ), 6460-6464). The transformation of the GPI-anchored prion protein to the scrapie protein isoform has also been associated with sphingolipid cholesterol microdomains (Taraboulos et al., J. Cell Biol. 129 ( 1995 ), 121-132). The gangliosides are therefore preferably used in such a way that the modulation of the sphingolipid cholesterol microdomains causes a change in the proteolysis of the amyloid precursor protein of Alzheimer's disease or a modification of a prion protein.

The modulation of sphingolipid cholesterol according to the invention Microdomains can be used for therapeutic, preventive as well diagnostic purposes may be of interest. The gangliosides are included preferably in a dose of 3, preferably from at least 5 to 30 and preferably administered up to 20 mg per kg per day.

The invention is illustrated by the following examples and the accompanying ones Figures explained further.

Fig. 1 shows that gangliosides inhibit the crosslinking of GPI-anchored proteins, in particular GH-DAF in living cells. MDCK GH-DAF cells (MDCK: dog, Cocker Spaniel, kidney) were treated with 0, 10, 50 and 100 μM GM ₁ (A) or with 10, 50 and 100 μM bbG (B) for 1 hour at 37 ° C. incubated. CHO FR-GPI cells were incubated with 100 μM bbG or with 100 μM GM ₁ for 1 hour at 37 ° C. The cells were then cross-linked with BS ³ . The proteins were separated with 5-15% SDS-PAGE and detected after Western blotting with an anti-GH antibody, followed by ECL (electrochemiluminescence). The autoradiographs were scanned and the intensity of the immunoreactive signal is given as the relative optical density (ROD) for 0 µM GM ₁ (A, upper curve), 0 µM bbG (B, upper curve), 100 µM GM ₁ (A, lower curve) and 100 µM bbG (B, lower curve) are shown.

Figure 2 shows that gangliosides inhibit cross-linking of FR-GPI in CHO cells. CHO FR-GPI cells were incubated with 100 μM bbG or with 100 μM GM ₁ for 1 hour at 37 ° C. The cells were then cross-linked with BS ³ . The proteins were separated with 5-15% SDS-PAGE and detected after Western blotting with an anti-folate receptor antibody, followed by ECL (electrochemiluminescence).

Figure 3 shows the incorporation of GM ₁ into the plasma membrane of MDCK GH-DAF cells. In (A) MDCK GH-DAF cells were incubated with GM ₁ containing 1 µCi / ml tritium at a final ganglioside concentration of 100 µM for 1 hour at 37 ° C and then with BSA (bovine serum albumin) for 0, 10, 20, Washed for 30 or 45 minutes (hatched bars). The additive effect of treatment with 0.1% trypsin for 5 minutes at 37 ° C after washing with BSA is shown as a solid bar. In (B) the crosslinking was carried out after incubation of GM _1- loaded cells with BSA for 0, 10, 20, 30 or 45 minutes. SDS-PAGE, Western blotting and detection were performed as described in Fig. 1.

Figure 4 shows the increase in detergent solubility of GH-DAF by gangliosides. In (A) MDCK GH-DAF cells with 100 µM bbG for 1 hour at 37 ° C or with 100 µM NBD-C ₅ -HPC for 1 hour at 8 ° C; Load with 100 µM GM ₁ for 1 hour at 37 ° C or with 100 µM bbG for 1 hour, followed by a 6 hour incubation with DMEM containing serum (A) or treatment with 10 mM methyl-β-cyclodextrin for 1 hour at 37 ° C or incubation with Anti-GH for 1 hour at 12 ° C, followed by incubation with a secondary antibody (B). The cells were extracted with Triton X-114 for 30 minutes at 4 ° C, centrifuged and the GH-DAF in the soluble (S) and insoluble (I) fraction was detected as described below.

Figure 5 shows that the effect of octyl glucoside on sphingolipid cholesterol microdomains is different from the effect of gangliosides. MDCK GH-DAF cells were incubated for 1 hour with 5 and 10 mM octyl glucoside and cross-linked with BS ³ . A slight decrease in cross-linking was only observed when using 10 mM octyl glucoside. The membranes remained intact at all concentrations examined.

Figure 6 shows that the inhibition of cross-linking of GH-DAF is a specific property of gangliosides. In (A) MDCK GH-DAF cells were incubated with 0, 10, 50 or 100 μM NBD-C ₆ -HPC for 30 minutes at 8 ° C. before they were cross-linked with BS ³ . SDS-PAGE, Western blotting and detection were performed as described in Fig. 1. In (B), MDCK GH-DAF cells were incubated with 0 or 100 µM bbG for 1 hour, followed by a 6 hour incubation with a medium containing serum (DMEM / FCS). The cells were cross-linked, followed by SDS-PAGE, Western blotting and detection as detailed in Figure 1.

Figure 7 shows that ganglioside treatment does not change the immunofluorescence pattern of GH-DAF. MDCK GH-DAF cells were incubated for 1 hour at 37 ° C. without (A, D), with 100 μM bbG (B) or with 10 mM CD (C). The cells were fixed and labeled with an anti-GH antibody followed by a Cy3-conjugated anti-sheep IgG (A, B and C) or crosslinked antibody-induced before fixation.

Figure 8 shows that loading MDCK GH-DAF cells with gangliosides changes the autophosphorylation status. MDCK GH-DAF cells were loaded with bbG or GM ₁ for 1 hour at 37 ° C or cross-linked with 0.5 mM BS ³ for 45 minutes at 4 ° C. After lysis, aliquots of the samples were separated on a 5-15% gel and the in-gel assay for protein kinase activity was performed. Bands in which the intensity increased during ganglioside treatment are marked with an arrow. Bands that were only visible after ganglioside loading are marked with an arrow head.

Fig. 9 shows the inhibition of the influenza HA and Copatching of GH-DAF by gangliosides. MDCK GH-DAF cells were infected with the influenza HA virus and then incubated with DMEM (AF) or 100 µM GM ₁ in DMEM (GL) for 1 hour at 37 ° C. After subsequent treatment with a mixture of monoclonal anti-HA mouse and polyclonal anti-GH sheep antibodies at 4 ° C, the patching was carried out using labeled secondary Cy-3 anti-sheep (red) - and FITC- Anti-mouse (green) - antibodies detected. The panels in the left column show the distribution of GH, in the middle column the distribution of HA and in the right column the combination of both signals. The panels DF each show a detail of the panels AC, whereas the panels JL show a detail of the panels GI. The lines in F and L correspond to 2 µM, those in C and I 8 µM.

Examples example 1 Inhibition of cross-linking of GPI-anchored proteins by exogenous Administration of gangliosides

To analyze the organization of GPI-anchored proteins on the surface, an MDCK cell line was used which permanently expresses GH fused to the GPI anchor of the decay accelerating factor (GH-DAF) (Friedrichson et al., Nature 394 ( 1998 ), 802-805). The MDCK GH-DAF cells were stored at 37 ° C. in 5% CO ₂ in DMEM, supplemented with 10% FCS and antibiotics. The experiments were carried out on confluent or sub-confluent cells grown in plastic switches. When MDCK GH-DAF cells were chemically cross-linked with bis (sulfosuccinimidyl) suberate (BS ³ ), a distinct band at 46 kDa (dimer) and a smeared band, which ranged from 60 kDa up to 300 kDa, were observed by Western blotting detected ( Fig. 1A).

To investigate the influence of gangliosides on sphingolipid-cholesterol rafts, MDCK GH-DAF cells were loaded with different concentrations of GM ₁ for 1 hour in a serum-free medium at 37 ° C and cross-linked with BS ³ . As can be seen in Figure 1A, treatment of MDCK GH-DAF cells with GM ₁ significantly reduced the amount of cross-linked GH-DAF. Quantification of the immunoreactive bands showed that 73% ± 6% of GH-DAF formed oligomers in untreated cells, while 65% ± 7%, 57% ± 7% and 49% ± 7% of GH-DAF in cross-linked species in cells found that were treated with 10, 50 and 100 µM GM ₁ . Incubation of the cells with bovine brain gangliosides (bbG) also efficiently inhibited the formation of GH-DAF oligomers ( Fig. 1B). When using 100 µM bbG, the cross-linking efficiency was 51% ± 5%, compared to 73% ± 6% in untreated cells.

To determine if the ability of gangliosides to dissolve rafts containing GPI-AP is independent of the cell type, CHO cells that permanently express the GPI-anchored folate receptor (FR-GPI) were treated in the same way. The CHO FR-GPI cells were grown in a folate-free Hams F-12 medium containing 5% FCS, hygromycin (100 µg / ml), penicillin (100 U / ml) and streptomycin (100 µg / ml). Cross-linking of CHO FR-GPI cells with BS ³ leads to the appearance of cross-linked products of FR-GPI, a cross-linking efficiency of 28% ± 3% being observed. When CHO FR-GPI cells were loaded with 100 µM GM ₁ or 100 µM bbG before cross-linking, the cross-linking efficiency was reduced to 2.7% ± 2.5% and 3.3% ± 2%, respectively (see Fig. 2 ).

In order to quantify the amount of GM ₁ that is incorporated into the plasma membrane, MDCK GH-DAF cells were incubated with GM ₁ containing 1 μCi / ml tritium at a final ganglioside concentration of 100 μM for 1 hour. For this, GM _{1 was} extracted, purified according to known methods (Tettamanti et al., Biochim. Biophys. Acta 296 ( 1973 ), 160-170) and radiolabeled on the C-3 of the long-chain base group (Sonnino et al., J. Lipid Res. 25 ( 1984 ), 620-629). The homogeneity of the radioactive compound ³ H-GM ₁ was over 99% and its specific radioactivity was 1.31 Ci / mmol. The cells were incubated for 1 hour in DMEM containing 1 µCi / ml of GM ₁ containing tritium at a final ganglioside concentration of 100 µM, washed with PBS / BSA and then incubated for 0 to 45 minutes with PBS / BSA at 37 ° C . The cells were then treated for 5 minutes at 37 ° C with 1 ml PBS containing 0.1% trypsin. The cell lysates were analyzed for radioactive ganglioside content with a liquid scintillation counter (Beckmann Instruments, CA). The protein was determined using a Protein Assay Reagent (BCA; Pierce, UK).

Only about 0.7% of the exogenously added ganglioside associated with the cells. About 35% of the total associated GM ₁ could be removed by incubating the cells with a BSA solution ( Fig. 3A). Subsequent treatment of the cells with trypsin revealed two pools of associated GM ₁ . About 30% of the remaining GM ₁ was released by trypsin treatment (trypsin-unstable pool) and about 70% was stable (trypsin-stable pool) ( Fig. 3A). It is believed that the trypsin-stable pool of GM ₁ corresponds to the molecules that are inserted into the plasma membrane, whereas the trypsin-labile pool consists of gangliosides that interact with proteins that protrude from the plasma membrane (cf. Masserini et al ., 1990 , Biochemistry 29, 697-701; Saqr et al., J. Neurochem. 61 ( 1993 ), 495-411). The trypsin-stable pool of GM ₁ contained the fraction which was resistant to extraction with Triton X-100 at 4 ° C. After incubating cells with GM ₁ containing 100 µM tritium, 3.74 nmoles GM ₁ (per mg protein) were found in the detergent-insoluble fraction, while 1.26 nmoles GM _{1 were obtained} from the soluble fraction, which shows that exogenous GM ₁ accumulates in the rafts.

In order to rule out that the inhibition of the crosslinking of GH-DAF takes place due to the action of gangliosides loosely associated with the surface, the crosslinking was carried out on GM _1- laden cells before and after the incubation with BSA. The crosslinking efficiency remained the same under these two conditions (see FIG. 3B, the variation in efficiency was less than 5% under all conditions). Thus gangliosides, which are firmly associated with the membrane, are responsible for the inhibition of cross-linking. It was found that 70% of the firmly associated GM _{1 was incorporated} into the bilayer according to the criterion used by other authors (Kanda et al., J. Biochem. (Tokyo) 91 ( 1982 ), 1707-1718; Schwarzmann et al. , Biochemistry 22 ( 1983 ), 5041-5048). The proportion of the trypsin-labile pool in cross-linking is therefore very small, if it occurs at all, especially since the solubility of GH-DAF in TX-114 has been increased considerably by gangliosides. This behavior cannot be explained by ganglioside micelles that bind to protruding proteins.

Example 2 Change in detergent solubility of GH-DAF by gangliosides

A frequently used criterion for examining the association of a protein with microdomains is its resistance to extraction against non-ionic detergents, such as Triton X-100 at 4 ° C (Brown et al., Cell 68 ( 1992 ), 533-544). The detergent solubility of GH-DAF was therefore investigated after loading the cells with ganglioside. It was found that the solubility of GH-DAF in TX-114 was significantly increased from 51 ± 4% to 83% ± 10% and 76% ± 8% in cells loaded with GM ₁ and bbG, respectively ( FIG. 4A). .

These results showed that gangliosides that are inserted into the plasma membrane displace GPI-APs from lipid microdomains. Influencing, in particular inhibition of cross-linking, by steric hindrance by gangliosides, which are bound to proteins protruding from the surface of the plasma membrane, thus does not occur or only to a very small extent. Reducing cholesterol in cell membranes is known to increase the solubility of GPI-APs in non-ionic detergents (Cerneus et al., J. Biol. Chem. 268 ( 1993 ), 3150-3155; Hanada et al., J Biol. Chem. 270 ( 1995 ), 6254-6260; Scheiffele et al., EMBO J. 16 ( 1997 ), 5501-5508). The solubility of GH-DAF was shifted to 80% ± 7% after extraction of the membrane cholesterol with CD in a TX-114 extraction ( FIG. 4B). In contrast, stabilization of the microdomains by antibody-induced cross-linking reduces the solubility of GH-DAF to 34% ± 7% ( Fig. 4B).

The results show that a modulation or change in the Lipid content of the cellular membrane by using exogenous Gangliosides both the cluster behavior of GPI-APs and the Solubilization modulated in TX-114 at 4 ° C.

Example 3 Effect of gangliosides on microdomains

As shown above, GPI-APs are displaced from the rafts by loading MDCK-GH-DAF cells with gangliosides. In order to rule out the possibility that this effect occurs in part due to the detergent-like properties of gangliosides, n-octyl-β-D-glucopyranoside (OG) was used. This detergent is chemically similar to gangliosides (it contains, for example, a carbohydrate head group) and is known to fully solubilize GPI-APs when used above the critical micelle concentration (20-25 mM). MDCK GH-DAF cells were incubated with different concentrations of OG and cross-linked with BS ³ . Using the same concentrations of OG as gangliosides (10-100 µM), no effect on cross-linking of GH-DAF was observed. Even when cells were incubated with a 100-fold higher concentration of OG (10 mM), only a slight inhibition of cross-linking was observed ( FIG. 5; the inhibition was 3.5% ± 0.7%). Remarkably, the membranes remained intact even under this condition, since caveolin-1, a protein membrane that has both N and C termini arranged towards the cytoplasm, was not cross-linked. Only when OG was used above its critical micelle concentration (20 mM) was caveolin-1 found to be a cross-linked, high molecular weight product. Treatment of MDCK GH-DAF cells with 100 µM GM ₁ did not lead to cross-linking of caveolin-1. These data show that the inhibition of GH-DAF cross-linking by gangliosides does not occur due to solubilization of the membrane by detergent-like properties of the gangliosides.

Example 4 Inhibition of cross-linking of GH-DAF with gangliosides

This example shows that the inhibition of clustering of GPI-anchored proteins is a specific property of gangliosides. A lipid that is not part of the sphingolipid-cholesterol microdomains should not have any influence on the cross-linking and the solubility of GH-DAF. Conveniently, phosphatidylcholine molecules should be used for such a control experiment, such as those that occur in cell membranes. However, these lipids cannot be introduced into the membrane by exogenous addition. For this purpose, MDCK GH-DAF cells were therefore incubated with a partially water-soluble fluorescence-labeled analogue of phosphatidylcholine (NBD-C ₆ -HPC). It is known that NBD-C ₆ lipids quickly reach the plasma membrane at low temperature and their accumulation in the plasma membrane can easily be observed by immunofluorescence microscopy (Kean et al., J. Cell. Biol. 123 ( 1993 ), 1403-1419 ). The detergent insolubility of NBD-C ₆ -HPC has now been investigated. Cells were loaded with NBD-C ₆ -HPC and detergent extraction was carried out at 4 ° C with Triton X-114. Over 99% of the NBD-C ₆ -HPC was recovered from the detergent-soluble fraction, indicating that the lipid is not associated with the microdomains. No change in the cross-linking pattern was observed after incubation with different concentrations of NBD-C ₆ -HPC. The cross-linking efficiency was 73% ± 5% in cells treated with 100 µM NBD-C ₆ -HPC compared to 73% ± 6% in control cells ( Fig. 6A). The solubility of GH-DAF (54% ± 2% soluble) was not changed by NBD-C ₆ -HPC ( Fig. 4A).

In order to determine whether the inhibition of the crosslinking of GH-DAF by gangliosides is reversible, MDCK GH-DAF cells were loaded with 100 μM bbG and then incubated for 6 hours in a medium containing serum before the crosslinking. As shown in Figure 6B, 6 hours of incubation with serum-containing medium was sufficient to almost completely restore the cross-linking pattern of GH-DAF. The cross-linking efficiency was 6% ± 1% lower than that of control cells.

The detergent solubility of GH-DAF in TX-114 was also restored by incubating bbG-loaded cells with serum-containing medium for 6 hours (61% ± 4% soluble) ( Fig. 4A).

This example shows that the inhibition of cross-linking of GH-DAF is specific and reversible with gangliosides.

Example 5 The distribution of GH-DAF is not changed by gangliosides

Sphingolipid cholesterol microdomains or rafts are small, highly dynamic structures that cannot be resolved by conventional microscopic techniques. It was investigated whether loading the cells with gangliosides leads to a global rearrangement of the plasma membrane and thus the GPI-APs. The distribution of GH-DAF on MDCK cells with or without bbG loading was analyzed by immunofluorescent labeling. In order to prevent a redistribution of GH-DAF after fixation, formaldehyde fixation was used in combination with subsequent methanol fixation (Harder et al., J. Cell Biol. 141 ( 1998 ), 929-942). Incubation of the cells with 100 μM bbG for 1 hour before fixation and antibody labeling had no influence on the diffuse distribution of GH-DAF on the cell surface ( FIGS. 7A and B). The intracellular staining comes from the arrangement of the protein in the Golgi and / or the endosomal arrangement. A reduction in membrane cholesterol with 10 mM CD also did not lead to any detectable changes in the distribution of GH-DAF ( FIG. 7C). In comparison, the addition of antibodies against GH-DAF prior to fixation resulted in a spotty distribution of the immunofluorescence signal ( FIG. 7D). These results show that the inhibition of cross-linking by gangliosides does not occur due to a rearrangement of the membrane.

Example 6 Loading the cells with gangliosides changes the Autophosphorylation state of protein kinases

This example shows that there is a correlation between the administration of gangliosides to MDCK GH-DAF cells and cell signaling processes. As an indication of signal transmission, the state of autophosphorylation of cell protein kinases after ganglioside loading of cells was measured using an in-gel assay. The conditions for the ganglioside loading were the same as for the cross-linking studies. As can be seen from Fig. 8, the intensity of various bands increased significantly after adding gangliosides (arrow), while other bands only became visible after ganglioside treatment (arrow heads). It should be noted here that chemical cross-linking with BS ³ alone does not lead to significant changes in the autophosphorylation state. These experiments show that gangliosides, in addition to modulating the association of GPI-anchored proteins on microdomains, can induce signaling processes in MDCK cells.

Example 8 Inhibition of copatching of influenza HA and GH-DAF by gangliosides

Sphingolipid cholesterol microdomains are small and highly dynamic structures that cannot be resolved by conventional microscopic techniques. It was investigated whether loading the cells with gangliosides leads to a redistribution of GPI-APs. The distribution of GH-DAF on MDCK cells with or without bbG loading was analyzed by immunofluorescent labeling. In order to prevent redistribution of GH-DAF after fixation, formaldehyde fixation was used in combination with subsequent methanol fixation (Harder et al., J. Cell. Biol. 141 ( 1998 ) 929-942). Incubation of the cells with 100 μM bbG for 1 hour before fixation and antibody labeling had no influence on the diffuse distribution of GH-DAF on the cell surface ( FIG. 8, panels A and B). The intracellular staining comes from the arrangement of the protein in the Goigi and / or the endosomal arrangement. A reduction in membrane cholesterol with 10 mM CD also did not lead to any detectable changes in the distribution of GH-DAF ( FIG. 7C). In comparison, the addition of antibodies against GH-DAF prior to fixation resulted in a patch-like distribution of the immunofluorescence signal ( FIG. 7D).

The simultaneous incubation of living cells with antibodies against The proteins associated with the microdomains lead to the redistribution of the independently cross-linked components in overlapping patches. On Copatching is only observed for raft marker pairs, not for pairs consisting of a raft marker and a non-raft marker, from which one can conclude that attractions in the common Lipid environment of raft proteins are present.  

It has now been investigated whether the exogenous administration of gangliosides disrupts the physical connection of GH-DAF to lipid microdomains. As the trimer became a protein marker associated with the microdomains Transmembrane protein influenza hemagglutinin (HA) in MDCK GH-DAF- Cells expressed.

The cells were incubated simultaneously with a monoclonal anti-HA mAb and a polyclonal anti-GH Ab at 4 ° C., followed by an incubation with the respective secondary antibodies. Microscopic analysis showed copatching of HA and GH-DAF in the majority of the cells (60% copatching, 35% partial overlap and 5% random distribution) ( Fig. 9A-F). When the cells were incubated for 1 hour with 100 µM GM ₁ before antibody-induced cross-linking, only partial cocustering of HA and GH-DAF was observed in most cells (31% cocustering, 50% partial overlap and 19% random distribution ) ( Fig. 9G-L). These results show that the attractive forces between patches of HA and GH-DAF are disrupted by administration of gangliosides, which shows that GPI-APs are displaced from the lipid microdomains.

Example 9 Experimental procedures

In the above examples, the following procedures were followed used:

Insertion of lipids, reduction of cholesterol and Octylglucoside treatment of cells:

Stock solutions of bovine brain gangliosides (bbG) or GM ₁ (1-10 mM) were prepared in PBS. The cells were loaded with 10-100 µM gangliosides in DMEM for 1 hour at 37 ° C (Masserini et al., Biochemistry 29 ( 1990 ), 697-701; Saqr et al., J. Neurochem. 61 ( 1993 ), 395 -411). To remove excess lipid, cells were washed thoroughly with PBS containing 2 mg / ml defatted BSA (PBS / BSA). An ethanolic 0.5% stock solution of 2- (6- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino) hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD -C ₆ -HPC) was injected into PBS with vigorous vortexing to give a 1 mM solution. The insertion of NBD-C ₆ -HPC in membranes was carried out with 10-100 μM NBD-C ₆ -HPC for 1 hour at 8 ° C. In order to rid MDCK GH-DAF cells of cholesterol, they were incubated for 1 hour at 37 ° C. with 10 mM methyl-β-cyclodextrin (CD). For the octyl glucoside treatment, MDCK GH-DAF cells were incubated for 1 hour at 37 ° C. with various concentrations of octyl glucoside dissolved in DMEM.

Detergent extraction

The cells were washed with cold PBS and with 1 ml TX-114 Lysis buffer extracted at 4 ° C for 30 minutes. The cells were scraped off and centrifuged samples for 30 minutes at 15,000 g at 4 ° C. The Supernatant (soluble fraction) was removed, the pellet (insoluble fraction) was resuspended in 1 ml lysis buffer. Soluble and insoluble fractions were precipitated on ice with 10% TCA for 1 hour and for 15 minutes Centrifuged at 15,000 g at 4 ° C. The pellets were washed with acetone (-20 ° C) washed and further processed with SDS-PAGE and Western blotting.

Cross-linking, electrophoresis and western blotting

The cross-linking protocol has been used as recently described (Friedrichson et al., Nature 394 ( 1998 ), 802-805). In short, cells were washed twice with cold PBS. Cross-linking was carried out with 0.5 mM BS ³ for 45 minutes at 4 ° C. Unreacted cross-linker was quenched with 50 mM glycine for 15 minutes at 4 ° C. The cells were cultured for 20 minutes at 4 ° C and 10 minutes at 37 ° C in a TX-114 lysis buffer (150mM NaCl, 10mM Tris-HCl, pH 8.0, 1mM EDTA, 1% Triton-X-114 and protease inhibitors). The lysates were briefly cooled on ice and clarified by centrifugation at 15,000 g for 15 minutes. The supernatants were subjected to a temperature-induced phase separation for 5 minutes at 37 ° C. Aqueous and detergent-enriched phases were separated by centrifugation for 3 minutes at 13,000 rpm at room temperature (RT). The aqueous phases were discarded and 0.9 ml of TX-114 wash buffer (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.06% Triton-X-114 and protease inhibitors) were added to the detergent phases . After centrifugation at 15,000 g at 4 ° C for 15 minutes, the samples were subjected to a further phase separation. Finally, the detergent phases were precipitated with cold acetone (-20 ° C) and boiled at 95 ° C for 5 minutes in a Lämmli sample buffer. The proteins were separated with a 5-15% SDS-PAGE and transferred to nitrocellulose. Polyclonal antibodies to GH and the folate receptor followed by the corresponding secondary antibodies and ECL were used to detect GH-DAF and FR-GPI. Immunoreactive bands were quantified by densitometric scanning of films or a LAS-1000 luminescence image analyzer (Fujifilm, Germany). The data for each condition was averaged and the change expressed as a standard deviation. The experiments were carried out 3 to 6 times. For some gels, the appropriate X-ray films were scanned using Photoshop software and the optical density of the immunoreactive bands was determined and printed out using MacBas (version 2.0 ).

Immunofluorescence

Cells grown on coverslips were washed 3 times with PBS and fixed for 6 minutes with 3.7% paraformaldehyde in PBS at 8 ° C and for 10 minutes with methanol at -20 ° C. The cells were then incubated for 30 minutes at room temperature with an anti-GH antibody in PBS, followed by incubation with Cy3-conjugated anti-sheep IgG for 30 minutes at room temperature. After each of the above incubations, three washes with PBS were performed. The coverslips were embedded in Moviol and images were taken with a high resolution digital camera C 4742-95 (Hamamatsu Photonics KK, Japan) and the digital deconvolution was carried out using the digital confocal routine Openlab (version 1.7.7 ) (Improvision, UK) .

For the antibody-induced cross-linking experiments, the Anti-GH antibody and the Cy3-conjugated anti-sheep IgG in DMEM diluted. The cells were washed with the anti-GH for 20 minutes at 37 ° C. Antibody incubated, washed with DMEM and for 20 minutes at 37 ° C incubated with Cy3-conjugated anti-sheep IgG. The cells were fixed and embedded in Moviol as described above.

In-gel protein kinase assay

Cells grown in 6-well dishes were loaded with gangliosides or cross-linked with BS ³ as described above, cooled on ice, washed 3 times with ice-cold PBS and in 400 μl lysis buffer (20 mM Tris acetate, pH 7.0, 0 , 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM β-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1% TX-100, 1 mM benzamidine, 2 µg / ml leupeptin, 0.1% β-mercaptoethanol , 0.27 M sucrose and 0.2 mM PMSF) were lysed for 30 minutes at 4 ° C. and scraped off with a rubber wiper. The lysates were clarified at 15,000 g for 15 minutes and aliquots were boiled with 5 × sample buffer for 5 minutes at 95 ° C. The samples were separated on 5-15% gels polymerized with 5 mg / ml BSA. The in-gel protein kinase assay was performed according to the method described by Dam (by Dam et al., EMBO J. 14 ( 1995 ), 1798-1811), except that 65 µCi γ- ³² P-ATP in 3 ml Buffer K per gel were used.

Viral infection and antibody-induced patching

For infection with the HA influenza virus, the virus was in an infection medium (MEM, 50 mM Hepes, pH 7.3, penicillin (100 U / ml) / streptomycin (100 µg / ml), 0.2% BSA) diluted and virus adsorption was carried out for 1 hour. The infection continued for another 2.5 hours. 100 µM GM ₁ diluted in DMEM was added to some cells over the past hour. For antibody-induced patching of HA and GH-DAF, the cells were incubated with a polyclonal anti-GH antibody and a monoclonal anti-HA-PR8 (H17L10) diluted 1: 3000 and 1:50 in DMEM. Incubation was carried out at 4 ° C for 1 hour. After a short wash with PBS / 0.2% BSA, the cells were incubated with the corresponding FITC and Cy-3-coupled secondary antibodies for 1 hour at 4 ° C. The cells were fixed, embedded and images were taken as described above. For the quantification, 60 randomly selected cells from two different experiments were saved as digital images. The cells were divided into three different categories according to the degree of overlap between patches: ( 1 ) cocluster (more than 70% overlap); ( 2 ) partial cocustering; ( 3 ) random distribution.

Claims (12)

1. Use of gangliosides for the preparation of an agent for Modulation of sphingolipid cholesterol microdomains. 2. Use according to claim 1, characterized, that the agent locates proteins on the sphingolipid Cholesterol microdomains affected. 3. Use according to claim 2, characterized, that the agent acylated the localization of anchor proteins Proteins, Src kinases and / or cholesterol-anchored proteins influenced. 4. Use according to claim 3, characterized, that means on glycosyl phosphatidylinositol anchor proteins, kinases the Src family, influenza virus hemagglutin and / or caveolin-1 in the sphingolipid cholesterol microdomain works. 5. Use according to one of the preceding claims, characterized, that the agent causes a cluster breakdown of the proteins. 6. Use according to one of the preceding claims, characterized in that the ganglioside is selected from bovine brain gangliosides, GM ₁ , GD1a, GD1b, GD3, GM2, GM3, GQ1a, GQ1b or / and globosides. 7. Use according to one of the preceding claims, characterized, that the modulation of the sphingolipid cholesterol microdomains is one Change in membrane transport, signal transmission and / or Cell adhesion properties and / or enzymatic processes causes. 8. Use according to one of the preceding claims, characterized, that the modulation of the sphingolipid cholesterol microdomains is one Change in proteolysis of the amyloid precursor protein Alzheimer's disease or a modification of a prion protein causes. 9. Use according to one of the preceding claims, characterized, that the agent is used as a therapeutic. 10. Use according to claim 10, characterized, that the agent in a dose of 3 mg to 30 mg per kg per day is administered. 11. Use of gangliosides to modulate Shingolipid Cholesterol microdomains. 12. Method for modulating shingolipid cholesterol microdomains, taking a patient gangliosides in a dose of 3 mg to 30 mg per kg per day.