DE102013005154B4 - Method for incorporation of lysosomal transmembrane proteins into cellular membranes - Google Patents

Abstract

The invention relates to a method for incorporating lysosomal transmembrane proteins into cellular membranes comprising the steps of: a) separating complete lysosomes from the cell, leaving the transmembrane proteins of the lysosome membranes structurally and functionally intact, b) preparing fusion liposomes with molecule types A, B, and C in which a cationic amphipathic molecule is selected for molecule type A, an aromatic molecule for molecule type B and a neutral helper lipid for molecule type C, c) preparation of fusogenic lyso-liposomes, wherein the lysosomes fuse with the fusion liposome, and d) fusion of the fusogenic lyso-liposomes with a cell membrane, the order between step a. and b. and in which the preparation of the fusion liposomes in step b. three types of molecules A: B: C from DOTAP: DiO: DOPE in a ratio of 1: 0.1: 1 mol / mol.

Description

The invention relates to a method for incorporation of lysosomal transmembrane proteins into cellular membranes.

State of the art

Lysosomes fulfill a large number of vital functions in living cells. Thus, they are centrally responsible for the degradation of biopolymers, which may be of non-cellular as well as cellular origin. The orderly breakdown of the lysosomes guarantees the presence of monomeric units for the construction of vital molecules as well as the maintenance of cellular homeostasis. The degradation within the lysosomes occurs via proteins associated with or in the membrane of lysosomes and activated under acidic conditions. Loss of functionality of individual lysosomal proteins can lead to the high accumulation of various substances in the cell and thereby trigger serious diseases. Examples are the lysosomal storage disease or Hunter's disease. Other hereditary heart diseases, such as Danon's Disease or a series of genetic hypertonic cardiomyopathies are mainly caused by the defect of the lysosomal transmembrane protein lysosomal-associated membrane protein 2 (LAMP2). The replacement of such transmembrane proteins by functional proteins would be of paramount importance as a therapeutic method, but is not routinely practicable with current methods.

According to the prior art, such proteins can be expressed by transfection into cells or first isolated after extensive purification procedures and then incorporated into the cells by the use of a carrier system.

By transferring foreign genetic material into a host cell (transfection), the formation of foreign proteins can be induced in them. There are many transfection techniques, from chemical to physical to biological. All function more or less well in the transfer of gene materials for the functional expression of cytoplasmic proteins, but only to a very unsatisfactory extent to generate functional transmembrane proteins. Although such proteins are disadvantageous in the host cell, they have lost functionality due to lack of modifications, incorrect localization or altered conformation, so that they can no longer act as a replacement for the original protein (Spampanato C., et al., Molecular Therapy (2011) 19 , 5, 860-869).

The second way to introduce foreign proteins into cellular membranes is based on purified proteins, which can be introduced into the membrane of target cells by fusogenic carrier systems.

Out US 2012/0183596 A1 For example, a method of introducing various negatively charged therapeutics into liposomes is known.

Out DE 10 2009 032 658 A1 For example, mixtures of amphipathic molecules and a method of cell membrane modification by fusion with these molecules are known.

Haaparanta, et al. (Haaparanta, T., Gustafsson, JA, Glaumann, H., (1983), Isolation of mitochondria, lysosomes, and microsomes from the rat anterior prostate with a note on inverted microsomal vesicles, Arch Biochem Biohys. 223 (2) 458- 67) a method for the isolation of lysosomes from rat cells is known.

From Csiszár et al. (Csiszár, A., Hersch, N., Dieluweit, S., Biehl, R., Merkel, R., Hoffmann, B., (2010), Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification, in: Bionconjugate Chem ., Pp. 537-543), another method for cell membrane modification by means of fusogenic liposomes is known.

The technical disadvantage of such methods is due to the great difficulties of protein purification of transmembrane proteins. Transmembrane proteins with several hydrophobic and hydrophilic domains undergo regular conformational changes and consequently a loss of functionality.

Object of the invention

The object of the invention is to introduce lysosomal membrane-bound proteins into living cells completely functional and in above-average amounts, thereby replacing missing or defective proteins in the membrane of lysosomes.

Solution of the task

The object is achieved by the method according to the main claim. Advantageous embodiments of this result in each case from the back-related claims.

Description of the invention

Lysosomal transmembrane proteins are used gently in their natural membrane environment without purification. To do this proteinaceous, lysosomal membrane fractions separated from cellular residues by simple physical separation techniques. In order to transfer the resulting membrane fractions, they are incorporated in a first step into a fusogenic, positively charged liposomal carrier system by membrane fusion, wherein the fusion of the lysosomal and liposomal membranes is ensured by the properties of the carrier. After the fusion of the two membrane fractions, the resulting lyso-liposomes are fused in a second step by contacting them with the plasma membrane of a living cell.

The minimum requirement for the method according to the invention is:
Positively charged fusion liposomes are used. They are z. B. from the WO 2011/003406 A2 are known and are referred to below as protein carriers. These include the molecule types A, B and C, wherein for molecule type A a cationic, amphipathic molecule, for molecule type B an aromatic molecule and for molecule type C a neutral helper lipid is chosen. The three types of molecules A: B: C from DOTAP: DiO: DOPE in the ratio 1: 0.1: 1 mol / mol are selected.

The lysosomal transmembrane proteins must be used in their natural cellular membrane environment. A separation of whole lysosomes from the cell takes place. The proteinaceous membrane fractions of whole lysosomes are prepared by simple physical separation methods such. As centrifugation, separated as efficiently as possible from the cellular residues. Thus, according to the invention, functionally and structurally complete lysosomal membranes are used.

In a first fusion step, the fusion liposome called the protein carrier and the lysosomal membrane fraction are brought into contact, and thereby fused. This fusion is preferably in an aqueous environment, e.g. B. at pH 7.4 and 37 ° C instead.

The second fusion then takes place between the lyso-liposomes prepared in the first step and the plasma membrane of the cell. This fusion is preferably carried out also in an aqueous environment, eg. At pH 7.4 and 37 ° C.

embodiments

Furthermore, the invention will be explained in more detail with reference to exemplary embodiments and the attached figures.

Show it:

1 Zeta potential distribution of lysosomes, fusion liposomes and lyso-liposomes.

2 CHO cells after treatment with fusion liposomes (FL / CHOs) and with fusogenic lyso-liposomes (Lyso-FL / CHOs). Measuring bar = 20 μm.

First step: separation of whole lysosomes from the cell

HeLa cells were cultured with FeDextran for 9 hours in a T175 culture flask. After changing the medium, the cells were kept in culture for a further 16 hours. In order to separate the paramagnetic FeDextan-enriched lysosomes from the remaining components of the cells, the cells were first homogenized by means of a 18G, then a 26G needle and then centrifuged at 4 ° C and 600 g. The supernatant was passed through a MidiMacs magnetic column. The lysosomes enriched in the paramagnetic FeDextran were retained by the magnetic field and thereby separated from the rest of the cells. After removal of the magnetic field, the structurally and functionally completely intact lysosomes were washed from the column with a buffer solution of 250 mM sugar with 4 mM imidazole / HCl (pH 7.4) and stored at -70 ° C. in the same buffer.

Second step: Preparation of fusion liposomes (protein carriers)

1,2-dioleoyl-3-trimethylammonium propane, chloride salt (DOTAP) as a positively charged lipid (type of molecule A), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a neutral (helper) lipid (Molecule C) and 3,3'-dioctadecyloxacarbocyanine Perchlorates (DiO) as fluorescence markers (Type B molecule) were dissolved in a chloroform / ethanol (10/1 v / v) solvent mixture in a molar ratio of DOTAP: DiO: DOPE of 1: 0.1: 1 mol / mol recorded. After mixing, the solution was dried in vacuo for 30-60 minutes and then resuspended in a buffer solution of 250 mM sugar with 4 mM imidazole / HCl (pH 7.4) to a final concentration of 2 mg lipid / ml buffer and sonicator 20 min homogenized. This stock solution could be used several times, provided it was stored at 4 ° C and homogenized again before use.

The fusion mixture is present as a liposome.

Third step: Fusion between lysosomes and fusion liposome production of fusogenic lyso-liposomes

The fusion liposome stock solution was diluted 1/20 to a lipid concentration of 0.1 mg / ml with the previous buffer solution. The lysosomes were also diluted 1/400 with the same buffer.

Since the particle concentration was not known in either solution, the optimal dilutions were adjusted by characterizing the zeta potential. Such a measurement will be in 1 shown. The peak area is proportional to the particle concentration and the peak position allowed the information of the zeta potential of the particles present. The zeta potential of lysosomes was measured between -25 and -19 mV, while the zeta potential of fusion liposomes was around +60 mV.

330 μl of the lysosomal sample were mixed with 660 μl of the fusion liposome stock solution (1: 2 v / v%) and fused for 10 min in a water bath at 37 ° C. After the fusion, the sample temperature was again cooled to 20 ° C and the zeta potential of the obtained fusogenic lyso-liposomes was determined ( 1 ). 1 shows that the zeta potential of the lyso liposomes lies, as expected, between the zeta potential of the lysosomes and that of the liposomes. Fusogenic lyso liposomes could be stored at 4 ° C for about 2 weeks without degradation.

Fourth step: Second fusion between the fusogenic lyso liposomes and a Chinese hamster ovarian cell line (CHO)

The prepared fusogenic lyso liposomes (200-300 μl) were pipetted to a cell culture dish with CHO cells (20,000-30,000 per dish, (d = 3.5 cm)) at 20 ° C room temperature and 37 ° for 5-10 minutes C merged. Subsequently, the cells were washed with the buffer used above. The fusion efficiency of the reagent with the cell membrane as well as the presence of the lysosomal LAMP2 protein were checked by fluorescence microscopy.

2 shows that effective and homogeneous membrane fusion has occurred between lyso-liposomes and the plasma membrane of the CHOs, as shown by the green-fluorescent membrane marker DiO (Figure D). The incorporation of LAMP2 lysosomal membrane protein exclusively into the membrane of the target cell was demonstrated by immunostaining with the anti-LAMP2-Cy3 antibody (see 2E ). 2 B above this shows no corresponding signals, since no fusogenic Lyso liposomes were used here.

The cells were gently fixed for this experiment, so that the antibody could not get into the cell but only to the membrane.

Claims (5)

Method for incorporating lysosomal transmembrane proteins into cellular membranes comprising the steps of: a. Separation of complete lysosomes from a cell, whereby the transmembrane proteins of the lysosome membranes remain structurally and functionally intact, b. Production of fusion liposomes with molecule types A, B and C, wherein for molecule type A a cationic, amphipathic molecule, for molecule type B an aromatic molecule and for molecule type C a neutral helper lipid is selected, c. bring the lysosomes into contact after step a. with fusion liposomes after step b. for producing fusogenic lyso-liposomes, whereby the lysosomes fuse with the fusion liposomes, and d. contacting the fusogenic lyso liposomes with a cell membrane whereby the lyso liposomes fuse with the cell membrane, the order between step a. and b. and in which the preparation of the fusion liposomes in step b. three types of molecules A: B: C from DOTAP: DiO: DOPE in a ratio of 1: 0.1: 1 mol / mol. A method according to claim 1, characterized in that the separation of the lysosomes from the cell takes place by means of physical separation. A method according to claim 1 or 2, characterized by carrying out a centrifugation as a separation step. Method according to one of the preceding claims, characterized in that for the production of fusogenic lyso liposomes, an approximately identical number of lysosomes and fusion liposomes are brought into contact with each other. Method according to one of the preceding claims, characterized in that membranes of mammalian cells are brought into contact with lyso-liposomes.

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