KR100565437B1 - Novel sphingomyelinase, the antibody against it, the antisense and the preparation method thereof - Google Patents

Abstract

The present invention relates to novel neutral sphingomyelinase derived from mammals, antibodies thereto, antisenses and methods for their preparation.

The novel neutral sphingomyelinase enzyme N-SMase ε isolated from the mammalian brain cells of the present invention is an HSP60 protein having a molecular weight of 60 kDa on SDS-PAGE, and has a dependent enzyme activity on magnesium ions. do.

N-SMase ε enzyme of the present invention can be used as a composition for the prevention and treatment of cancer diseases caused by abnormal cell proliferation by inducing apoptosis by generating ceramide.

In addition, the antibody of N-SMase ε of the present invention inhibits the activity of the enzyme, and the antisense of N-SMase ε inhibits the expression and activation of the enzyme, thereby inhibiting the production of ceramide, thereby inducing cell death, aging, and inflammation caused by ceramide. It can be used as a medicine for diseases.

Sphingomyelinase, ceramide, antibody, antisense, apoptosis, aging, inflammation

Description

Novel sphingomyelinase, the antibody against it, the antisense and the preparation method             

1 is a two-dimensional electrophoretic protein profile of purified active fractions from bovine brain,

Figure 2 is a graph showing the effect on sphingomyelin (SM) and ceramide of the cerebral cortical neural membrane of the purified N-SMase ε of the present invention,

Figure 3 is a graph showing the activity of GSH, DTT and 2-mercaptoethanol of the purified N-SMase ε of the present invention,

Figure 4 is a diagram observing the distribution in the tissue of the N-SMase ε of the present invention,

5 is a diagram illustrating the distribution of N-SMase ε in brain neurons and non-neuronal cells of the present invention,

6 is a graph showing the change in activity of N-SMase ε of the present invention by the HSP60 antibody obtained in rats,

7 is a photograph analyzing the N-SMase ε immunoprecipitated by beads and HSP60 antibody obtained from the rat,

8 is a secondary electrophoresis and silver stained picture of the HSP60 antibody and immunoprecipitated protein,

9 is a photograph of the immunoblotting secondary electrophoresis of the HSP60 antibody and immunoprecipitated protein,

10 is a graph measuring HSP60 protein expression and SMase activity by infecting PCDNA3-HSP60 with a cell line,

11 is a graph measuring HSP60 protein expression and SMase activity by treating sense or antisense to a cell line infected with PCDNA3-HSP60.

12 is a graph of siRNA treatment of PCDNA3-HSP60-infected cell lines to measure HSP60 protein expression and SMase activity.

Figure 13 is a form of normal cortical neurons and TNF-α-treated cortical neurons,

14 is a graph measuring specific activity of N-SMase ε at different times in TNF-α-treated cerebral cortical neurons,

15 is a result of immunoblot analysis using N-SMase ε antibody of the present invention in cerebral cortical neurons treated with TNF-α,

FIG. 16 is a diagram illustrating the quantitative change of ceramide and sphingomyelin and the effect of FB1, a ceramide production inhibitor, in cerebral cortical neurons treated with TNF-α. FIG.

The present invention relates to a novel enzyme derived from a mammal and a method for isolating and purifying the same, and in particular, isolating and purifying a novel sphingomyelinase and comprising an antibody and antisense prepared thereon. It relates to a composition for the prevention and treatment of diseases caused by ceramide.

Hydrolysis of sphingomyelin (SM), known as the SM pathway, is induced by the activation of sphingomyelinase (SMase), which produces a secondary messenger, ceramide. In doing so, ceramides play an important role in cellular responses such as apoptosis, differentiation, aging and inflammation.

Many recent studies have been carried out on the enzyme Neutral-sphingomyelinase (N-SMase), which plays a role in the production of the signal carrier ceramide (Hannun, YA; Science 274 , pp 1855-1859, 1996 ). However, their biochemical properties as well as the primary structures of the enzymes are little known. Therefore, present identification and characterization of SMases is an important cornerstone for elucidating the mechanism by which ceramide is produced and its exact intracellular role.

Some N-SMase activities have been described in mammalian brain (Maruyama, EN & Arima, M .; J. Neurochem . 52 , pp611-618, 1989; Liu, B., et al .; J. Biol) Chem . 273, pp34472-34479, 1998; Bernardo, K., et al . ; J. Biol. Chem . 275 , pp7641-7647, 2000; Jung, SY, et al .; J. Neurochem . 75 , pp1004- 1014, 2000) and N-SMase 1 (Tomiuk, S., et al . ; Proc. Natl. Acad. Sci. USA 95 , pp3638-3643, 1998) and 71 kDa N-SMase 2 (Hofmann, K., et al. Proc. Natl. Acad. Sci. USA 97 , pp5895-5900, 2000) has been cloned and characterized using bioinformatics based gene discovery methods. N-SMase 1 was more identified as lyso-platelet activating factor (Lyso-PAF) phospholipase C (PLC) than SMase (Sawai, H., et al . ; J. Biol. Chem. 274 , pp38131-38139 , 1999), N-SMase 2 activity was not inhibited by 10 mM glutathione (GSH), which is known to inhibit most of the previously described N-SMases. Therefore, N-SMase has not been identified as a signal-coupled effector that produces ceramides.

TNF-α is known as a neurotoxin candidate that plays a role in the progression of brain disease (Robertson, J., et al . ; J. Cell. Biol. 155 , pp217-226, 2001; Talley, AK, et. al .; Mol. Cell. Biol. 15 , pp2359-66, 1995; Sairanen, TR, et al . ; J. Neurol. Sci . 186 , pp87-99, 2001), its mechanism is not well known. Recent evidence shows that N-SMase plays an important role in apoptosis-induced signaling by TNF-α. Inhibitors of N-SMase efficiently inhibit TNF-α-induced apoptosis in MCF cells (Luberto, C., J. Biol. Chem. 277, pp41128-41139, 2002), which is an N-SMase mediated TNF- It suggests that α-signaling may be present.

We have previously disclosed various forms of membrane-associated N-SMase isolated from bovine and human brains (Jung, SY, et al .; Identification of multiple forms of membrane-associated neutral sphingomyelinase in bovine brain. J. Neurochem . 75 , pp 1004-1014, 2000).

Therefore, the present inventors conducted a study to isolate a new SMase that produces ceramides associated with many diseases. As a result, Mg ²⁺ -dependent and neutral 60 kDa new SMase is isolated and purified from mammalian cow brain. And antisense was prepared, and confirmed the mediation of TNF-α-induced apoptosis (TNF-α-induced apoptosis) to complete the present invention.

The present invention provides a novel sphingomyelinase and seeks to provide an effective anticancer or anti-inflammatory agent that can produce antibodies, antisenses thereof to modulate the production of ceramides associated with apoptosis and inflammation.

To achieve the above object, the present invention provides a Mg ^{2+ -dependent} , membrane-associated, neutral 60 kDa SMase derived from mammalian cells.

Sphingomyelinase (SMase) is an enzyme that acts on sphingomyelin (hereinafter SM) to produce ceramide, and is named N-SMase ε for the novel SMase of the present invention.

The present invention also provides a method for isolating and purifying N-SMase ε from mammalian cells.

Hereinafter, the present invention will be described in detail.

For example, N-SMase ε of the present invention is homogenized by mixing the bovine brain with homogenization buffer and centrifuged to remove the debris and nuclei of the cells, and then centrifuged to pellet the pellet obtained by ammonium sulfate-containing buffer 1 step of obtaining an enzyme source 'ammonium sulfate extract' for refining neutral SMase ε by resuspending and stirring in the mixture;

A second step of passing the ammonium sulfate extract of step 1 through a DEAE-cellulose column and eluting with a buffer containing ammonium sulfate and Triton X-100 to obtain an active fraction;

A third step of passing the active fraction of step 2 through a butyl-toyopearl column and eluting with tertiary distilled water to obtain an active fraction;

A fourth step of passing the active fraction of step 3 through DEAE-5PW HPLC and eluting with a buffer containing ammonium sulfate and Triton X-100 to obtain an active fraction;

A fifth step of passing the active fraction of step 4 through phenyl-5PW HPLC and eluting with tertiary distilled water to obtain an active fraction;

It is possible to separate and purify from the manufacturing method comprising a sixth step of passing the active fraction of step 5 through a mono S cation exchange HPLC and eluting with a buffer containing sodium chloride and Triton X-100 to obtain an active fraction. Do.

Finally, the neutral SMase ε of the present invention was purified about 38,000 times through 6 chromatographic steps.

The SMase ε of the present invention purified through the mono S cation exchange HPLC was separated into four spots in a two-dimensional SDS-PAGE, and the 60 kDa protein spot on the gel was identified as HSP60 through MALDI-TOF and Swiss plot database investigation. Confirmed. It can also be confirmed that HSP60 is N-SMase ε by also observing an increase in SMase activity in HSP60 expressing cell lines.

The Lineweaver-Burk plot for purified N-SMase ε is shown in a straight line with Km of 47 μM and Vmax of> 4,800 nmol / min / mg.

SMase ε of the present invention shows a substrate specificity 50 times higher than sphingomyelin than other phospholipids as a substrate, the enzyme activity is dependent on magnesium ions, and is expressed in nerve cells of the brain.

Enzyme activity of N-SMase ε increases until the concentration of glutathione is 3 mM, but gradually decreases and is completely inhibited at 10 mM, and the enzyme activity is unchanged for reducing agents such as DTT and 2-mercaptoethanol.

The present invention provides a neutral SMase bound to new cell membranes derived from mammalian cells, and the present invention also homogenizes mammalian brain cells and solubilizes them with ammonium sulfate or Triton X-100, followed by hydrophobic, ion exchange chromatography. By performing the chromatography and the like to provide an active fraction having the activity of the membrane-bound neutral SMase.

TNF-α treatment increases the amount of N-SMase expressing protein, thereby increasing specific activity to hydrolyze sphingomyelin to produce ceramide, which is not affected by fumonisin B1 (FB1). .

N-SMase ε mediates TNF-α-induced neuronal cell death through ceramide production.

The present invention provides an apoptosis activator containing N-SMase ε enzyme.

The present invention also provides a composition for inhibiting, preventing and treating the onset of cancer disease due to abnormal cell proliferation containing N-SMase ε enzyme.

The present invention provides the use of the N-SMase ε enzyme for the preparation of a therapeutic agent for cancer diseases caused by abnormal cell proliferation.

In addition, the gene expressing N-SMase ε can be used to treat and improve cancer diseases by increasing the production of ceramide by inhibiting the proliferation of cancer cells.

The cancer diseases include lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer, breast cancer, and fallopian tube carcinoma , Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer , Chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma, etc. It includes.

The present invention also provides an antisense that inhibits the expression of the N-SMase ε and N-SMase ε.

The N-SMase ε antibody of the present invention may specifically bind to the N-SMase ε of the present invention, that is, HSP60.

The N-SMase ε antibody of the present invention can be prepared by separating from a gel with active fractions obtained from phenyl-5PW HPLC as a antigen during the purification step, mixing with an adjuvant and injecting the mouse, and collecting serum from the mouse. Can be. This can prepare a mouse anti-60kDa protein antiserum, from which two monoclonal antibodies, SM1A5 and SM1E1, can be prepared.

Antisense that inhibits the expression of N-SMase ε can be designed by a conventional method in the art as a sequence for inhibiting the mRNA expression of hsp60, preferably A-ODN 5'-AGCATTTCTGCGGGG-3 'of SEQ ID NO: 3 , Si-HSP60 sense 5'-UGCUCACCGUAAGCCUUUGdTdT-3 'of SEQ ID NO: 5 and si-HSP60 antisense 5'-dTdTACGAGUGGCAUUCGGAAAC-3' of SEQ ID NO: 6.

Treatment of the N-SMase antibody, HSP60 antibody or hsp60 siRNA of the present invention may reduce the expression and activity of the N-SMase ε enzyme, thereby reducing the production of ceramide and ultimately preventing or preventing diseases caused by ceramide. It can be used to treat.

The present invention also provides a composition for preventing and treating inflammatory diseases caused by ceramides containing an antibody of N-SMase ε or an antisense of N-SMase ε as an active ingredient.

The present invention provides the use of N-SMase ε antibody or antisense of N-SMase ε for the preparation of a therapeutic agent for inflammatory diseases caused by ceramide.

In addition, the antibody or antisense of the N-SMase ε enzyme, N-SMase ε of the present invention can be usefully used for the study of the mechanism of cancer disease or inflammatory disease.

For example, if the expression, function, and regulatory mechanism of the N-SMase ε enzyme of the present invention is revealed, the pathway of action of metabolites by N-SMase ε enzyme and the mechanism of occurrence of diseases in which they are involved may also be revealed. In addition, by mutating the N-SMase ε enzyme of the present invention to lower or inactivate activity, it is possible to diagnose, prevent and treat diseases involving N-SMase ε enzyme.

In addition, the N-SMase ε enzyme of the present invention can be usefully used for the study of physiological action by the enzyme and ceramide.

The composition containing the antisense of N-SMase ε or N-SMase ε antibody or N-SMase ε of the present invention as an active ingredient can be added together with a substance or derivative having similar functions as the active ingredient, and Depending on the composition, it may further contain other active ingredients.

The composition containing the antisense of N-SMase ε or N-SMase ε antibody or N-SMase ε of the present invention as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. have.

Carriers, excipients, and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The compositions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. have.

The amount of the composition of the present invention may vary depending on the age, sex, and weight of the patient, but the amount of 0.001 to 100 mg / kg may be administered once to several times per week. Dosage may also be increased or decreased depending on the route of administration, extent of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The composition of the present invention can be administered by a route selected from oral, parenteral (including percutaneous, intradermal, intramuscular and intravenous), rectal and inhalation, preferably by intramuscular injection.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. The following examples and experimental examples are illustrative of the present invention, but the content of the present invention is not limited thereto.

Reference Example 1. N-SMase Activity Assay

The enzyme substrate [N-methyl- ¹⁴ C] SM (labeled at the choline site at ¹⁴ C) was dried under nitrogen and resuspended in ethanol.

Standard culture systems (100 μM) for N-SMase activity assays include 10 mM MgSO ₄ , 50 mM [N-methyl- ¹⁴ C] SM (approximately 60,000 cpm), 2 mM sodium deoxycholate (SDC) and 100 It was prepared to contain mM Tris-HCl, pH 7.0. According to the method of Bligh and Dyer (Bligh, EG & Dyer, WA; J. Can. J. Biochem. Biophysiol. , 37 , pp680-685, 1970), the reaction was carried out at 37 ° C. for 30 minutes and discharged by SMase. Extracted [N-methyl- ¹⁴ C] phosphocholine. The reaction was stopped by adding 320 μl of chloroform / methanol (1: 1, volume ratio) and 30 μl of 2N-HCl to the reaction mixture. After vortexing, the mixture was centrifuged to separate into two phases. 200 μl of clear solution layer was added to 1.2 ml of scintillation solution (Insta gel-XF, Packard Instrument Co., Meriden, CT, USA) and Packard Tri-carb liquid β-scintillation counter Radioactivity was measured with). Active fractions were collected and used in the next step.

To further define enzymatic activity as SMase, [ ³ H] ceramide produced from [ ³ H] SM of cerebral cortical neurons of rats labeled with [ ³ H] serine as substrate was measured. To label sphingolipid, neurons (1 × 10 ⁶ cells) were labeled for 72 hours in the presence of 1.0 μCi / ml [ ³ H] serine and washed with Tris-buffered saline. Cells were lysed and lipids were extracted with chloroform: methanol: 2N-HCl (100: 100: 1, v / v / v). Total lipids were dried and resuspended in ethanol. Lipids were used as substrates for testing the activity of SMase by analyzing SM degradation and ceramide production. The SMase activity test system (100 μl) contained the active fraction of the final mono S column purified from bovine brain as an enzyme source, 100 mM Tris-HCl, pH 7.0, 5 mM MgCl ₂ , 2 mM sodium deoxycholate and substrate (18,520). cpm). The reaction was stopped by incubating at 37 ° C. for 1 hour according to the method of Blywaer and then adding 320 μl of chloroform: methanol (1: 1, v / v) and 30 μl of 2N-HCl.

The mixture was separated into two phases by vortex and centrifugation. The organic layer was dried to remove glycerphospholipid through weak alkaline hydrolysis such as 0.1 M methanolic KOH for 1 hour at 37 ° C. The resulting lipid extract was spotted on a TLC silica plate and developed to the top of the plate in chloroform: methanol: acetic acid: water (85.0: 4.5: 5.0: 0.5, v / v / v / v), which is a good condition for the movement of ceramide. The plate was redeployed again in chloroform: methanol: acetic acid: water (65.0: 25.0: 8.8: 4.5, v / v / v / v). Each lipid was visualized by Iodine vapor staining. Radioactive spots corresponding to SM and ceramide were scraped off and measured with a β-liquid scintillation counter.

Example 1. Membrane Association, Mg from Bovine Brain ²⁺ -Dependent purification of N-SMase (N-SMase ε)

1-1. Preparation of Ammonium Sulfate Extract (Step 1)

N-SMase ε, a salt extractable form of membrane-bound N-mSMase, was purified as follows.

First, in order to prepare an enzyme source for the purification of N-SMase, the fresh bovine brain (5Kg) stored at -70 ℃ 5 times (25 liters) homogenizing buffer V (50mM Tris-HCl , pH 7.5, 1 mM EDTA, 3 mM MgCl ₂ , 50 mM KCl and 10 mM 2-mercaptoethanol) and homogenized with a polytron homogenizer (Model PT-MR 6000, Kinematica, Switzerland). Homogenates were centrifuged at 10,000 X g for 10 minutes to remove debris and nuclei. The supernatant was again centrifuged at 10,000 X g for 1 hour at 4 ° C. The pellet obtained by this 10,000 X g centrifugation was resuspended in 2.5 liter homogenate V and centrifuged at 40,000 X g for 1 hour. The pellet obtained by this 40,000 X g centrifugation was resuspended in 2.5 liter buffer V adjusted to 0.5 M (NH ₄ ) ₂ SO ₄ , and after stirring for 1 hour at 4 ° C, 40,000 X Centrifuged at g for 1 hour. The resulting supernatant was named 'ammonium sulfate extract' and was collected and used as an enzyme source for the purification of N-SMase ε.

1-2. DEAE-cellulose column chromatography (step 2)

The ammonium sulfate extract (2.5 liters) of step 1-1 was a DEAE-cellulose column (7 × 45 cm, bed, previously equilibrated with buffer D (25 mM Tris-HCl, pH 7.5, 1 mM EDTA and 10 mM 2-mercaptoethanol) volume, 2.0 liters). In this process, anything showing SMase activity effectively binds to the column. The protein bound to the column was sequentially eluted with Buffer D containing 0.5M (NH ₄ ) ₂ SO ₄ and 0.1% Triton X-100 at a flow rate of 20 ml / min. 10 μl of each fraction (40 mL) was used to analyze N-mSMase activity.

1-3. Butyl-Toyopearl Column Chromatography (Step 3)

The active fractions were collected and sonicated at 4 ° C. at 6 ° C. and 5 seconds at 3 ° C. using a cell disruptor (Sonics & Materials Inc., Danbury, CT, USA) for 1 hour at 4 ° C. and 100,000 X g. Centrifugation. An aliquot of a stock solution of 4.0M (NH ₄ ) ₂ SO ₄ was added to the supernatant obtained above to adjust to 0.5M (NH ₄ ) ₂ SO ₄ . Samples were run on a butyl-Toyopearl column, bed volume 150 mL, previously equilibrated with buffer D containing 0.5 M (NH ₄ ) ₂ SO ₄ .

The protein bound to the column was eluted continuously and sequentially with buffer D containing 0.2 M (NH ₄ ) ₂ SO ₄ at a flow rate of 15 mL / min and eluted again with distilled water. Aliquots (10 μl) of each fraction (30 mL) were tested for N-SMase activation measurements.

1-4. DEAE-5PW HPLC (Step 4)

The active fractions were collected and developed on a DEAE-5PW HPLC column (21.5 mm X 15 cm, Tosoh Co., Tokyo, Japan) previously equilibrated with Buffer D.

The protein bound to the column was eluted sequentially with a 2000 mL-linear concentration gradient of buffer D containing 0.5 M (NH ₄ ) ₂ SO ₄ and 0.1% Triton X-100 at a flow rate of 5 mL / min. . Aliquots (3 μl) of each fraction (5 mL) were tested for N-mSMase activity assay.

1-5. Phenyl-5PW HPLC (Step 5)

The active fractions were collected and applied to a Phenyl-5PW HPLC column (21.5 mm X 15 cm, Tosoh Co., Tokyo, Japan) pre-equilibrated with Buffer D containing 0.2 M (NH ₄ ) ₂ SO ₄ . The protein bound to the column was eluted with a 2000 mL-gradient with distilled water at a flow rate of 5 mL / min.

1-6. Mono S Cation Exchange FPLC (Step 6)

The active pool is a Mono S cation exchange FPLC column (5.0 cm X 5.0 mm, Pharmacia LKB Co., Pre-Equilibrated with Buffer S, mixed with an equal volume of Buffer S (25 mM sodium acetate, pH 6.5, 1 mM EDTA). Uppsala, Sweden).

The protein bound to the column was sequentially sequential with a linear concentration gradient of 20 ml of 0.0M to 1.0M NaCl and a gradual concentration gradient of Buffer S containing 1.0M NaCl and 0.1% Triton X-100 at a flow rate of 1 ml / min. Eluted. Aliquots (3 μl) of each fraction (1 mL) were tested for N-mSMase activity measurements.

N-SMase ε was purified almost homogeneously from the salt extract of bovine brain membrane, yielded 1.3%, and purified about 38,000-fold through six chromatographic steps.

Example 2. Characterization of Purified SMase ε

2-1. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The first denaturing SDS-PAGE was 10% polyacrylamide in the Biorad electrophoresis system (Protean II, Bio-Rad) according to Laemli's method (Laemmli, UK; Nature , 227 , pp680-685, 1970). It was performed on a gel. Secondary gel electrophoresis was made using the IPG-phor (Amersham Pharmacia Biotech, Uppsala, Sweden) system according to O'Farrell, PH; J. BIol. Chem. , 250 , pp4007-4021, 1975. Was performed according to the method. The isolated proteins were stained with PlusOne Silver staining kit (Pharmacia Biotech, Piscataway, NJ).

Each aliquot (10 μl) of the active fractions from the mono S column of steps 1-6 of Example 1 was mixed with an aliquot of Laemry sample buffer and 0.125M Tris-HCl, pH 6.8, 4% SDS, 20 % Glycerol, and 0.002% bromophenol blue. After boiling for 5 minutes, the samples were cooled to room temperature, and then subjected to 10% polyacrylamide gel electrophoresis. The isolated proteins were stained with a staining kit (Pharmacia LKB Co., Uppsala, Sweden).

Although most of the active fractions at the end of the purification process were separated into four spots in the two-dimensional electrophoretic protein profile, the total protein amount was less than 1 μg (see FIG. 1).

2-2. Protein Identification by Peptide Mass Fingerprinting Assay

Protein peptide fingerprinting analysis was performed according to the method of Park et al . (Park, JB et al . ; J. Biol. Chem. , 275 , pp21295-21301, 2000).

In summary, 42 kDa spots were stained with Coomassie Brilliant Blue, cut off from secondary electrophoresis gels and treated with trypsin. An aliquot of 1 μl in the reaction solution (30 μl total volume) was used for peptide mass fingerprinting. The mass of the tryptic peptide was measured with a mass spectrometer (Bruker Reflex III). Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) analysis was performed using α-cyano-4-hydroxycinnamic acid as a matrix. The peak of the trypsin autolysis product was used for internal calibration. Peptides were identified using a comparison of mass values against the SWISS-PROT database.

As a result of MALDI-TOF mass spectrometry and SWISS PROT database investigation, all four spots of 60 kDa purified through the six steps of purification and 60 kDa of immunoprecipitates were identified as HSP60.

2-3. Enzyme activity

Enzyme Kinetics is a method for calculating Michaelis constant Km, limit velocity Vmax, and rate in a reaction in which an enzyme acts as a catalyst, based on the Michaelis Menton equation.

The Lineweaver-Burk plot of the purified N-SMase ε is shown in a straight line with a Km of 47 μM and a Vmax of> 4,800 nmol / min / mg.

2-4. Substrate specificity

The substrate specificity of the enzyme was determined using various phospholipids as substrates. This enzyme i.e. the various substrates, N- [methyl - ¹⁴ C] sphingomyelin ^{(N- [methyl- 14 C] sphingomyelin} ) (SM), 1,2- di-palmitoyl-3-phosphatidyl [N- methyl ^-3 H] choline (1,2-dipalmitoyl-3-phosphatidyl [N-mehtyl- ³ H] choline) (DPPC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho [2- ³ H] inositol (1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho [2- ³ H] inositol) (PI), 1,2-dioleoyl-3-phosphatidyl [3- ¹⁴ C] serine (1,2-dioleoyl-3-phosphatidyl [3- ¹⁴ C] serine) (PS), 1-stearoyl-2- [1- ¹⁴ C] arachidonoyl-GPC (1-stearoyl-2 ^{- [1- 14 C] arachidonoyl-} GPC) (2-AA-PC), 1- acyl -2- [1- ¹⁴ C] Araki FIG Russo -GPE (1-acyl-2- [ 1- 14 C] arachidonoyl -GPE) (2-AA-PE) showed at least 50-fold higher specificity for SM.

Purified N-SMase ε was incubated with cortical neuronal membranes metabolically biosynthesized from [ ³ H] serine. SM decreased while ceramide increased (see FIG. 2).

2-5. Effect by Glutathione, DTT, β-mercaptoethanol

GSH is known to act as a possible regulator of N-SMase in a variety of cells during cell damage and apoptosis, regardless of the deactivation by the sulfhydryl moiety. That is, intracellular reduction of GSH suggests increased N-SMase activity. When testing N-SMase ε for the effect by GSH, two types of profiles were observed. Enzyme activity was increased up to 3 mM, but gradually decreased and completely inhibited at 10 mM. On the other hand, like other reducing agents, DTT and 2-mercaptoethanol had no effect (see Fig. 3). These results suggest that, among the N-SMase enzymes tested for GSH effects, N-SMase ε may be the most sensitive to the reduction of intracellular GSH.

2-6. Effect of various cations

N-SMase ε absolutely requires Mg ²⁺ ions in the test assay, and the activity of this enzyme stimulated by Mg ²⁺ was influenced by other cations. Ca ²⁺ had no effect until it reached 0.5 mM, but Cu ²⁺ , Zn ²⁺ and Fe ²⁺ strongly inhibited the activity, resulting in IC ₅₀ of 28 μM, 32 μM, and 47 μM, respectively. In contrast, Fe ³⁺ increased the activity by 1.8 times. In particular, the regulation of enzyme activity by Fe has been shown to be very important for the production of ceramides and other continuous metabolites under various oxidative stresses. This is because the conversion of Fe ²⁺ to Fe ³⁺ in such an environment activates N-SMase ε with a decrease in GSH.

2-7. Influence by Cyposstatin

The N-SMase activity fraction was pre-reacted with cypressstatin at 37 ° C. for 10 minutes, and then reacted for 30 minutes by adding 50 mM [N-methyl- ¹⁴ C] SM (about 60,000 cpm). [N-Methyl- ¹⁴ C] phosphocholine extracted by SMase was extracted according to the method of Blae and Dyer. The reaction was stopped by adding 320 μl of chloroform / methanol (1: 1, volume ratio) and 30 μl of 2N-HCl to the reaction mixture. After vortexing, the mixture was centrifuged to separate into two phases. 200 μl of clear solution layer was added to 1.2 ml of scintillation solution (Insta gel-XF, Packard Instrument Co., Meriden, CT, USA) and Packard Tri-carb liquid β-scintillation counter Radioactivity was measured with).

N-SMase ε activity can be inhibited by cyphostatin, which is a known inhibitor of N-SMase and Ki is 50 μM.

2-8. Tissue and Intracellular Distribution of N-SMase ε

To test the tissue and intracellular distribution of N-SMase ε in the brain, we used non-neuronal cells as tissues of the rat (liver, splin, intestine, colon, heart, lung, brain and kidney) and neurons, And glia were prepared and analyzed by immunoblotting using the anti-rHSP60 antibody prepared in Example 3 below.

N-SMase ε was distributed in large numbers in the brain (see FIG. 4) and was almost entirely present in neurons rather than glia (see FIG. 5). This suggests that N-SMase ε plays an important role in neuronal action. This suggests that ceramides are involved in various functions of cells, including apoptosis, for various types of neurons.

Example 3. Preparation and Identification of Antibodies

3-1. Preparation of 60kDa Protein N-SMase ε Monoclonal Antibody

To generate antisera of anti-60kDa protein from mice, 60kDa protein was isolated from a gel with active fractions obtained from phenyl-5PW HPLC, Example 1, steps 1-5 of Example 1 as an antigen, and then mixed with adjuvant. Serum was collected from mice after injection into mice. Mouse anti-60kDa protein antiserum was prepared and two monoclonal antibodies, SM1A5 and SM1E1, were prepared.

3-2. rHSP60 Antibody Preparation

The cDNA encoding it was cloned by using anti-60kDa protein antiserum from rat brain λ ZAP library and the 60kDa protein was identified as HSP60. In addition, to confirm that HSP60 has N-SMase ε activity, first, human HSP60 was overexpressed in E. coli and purified from inclusion bodies. Polyclonal anti-recombinant HSP60 (rHSP60) antibodies were then generated from the rats and the antibodies were purified from rHSP60-immobilized Protein G affinity column.

Specifically, human HSP60 cDNA was amplified with primer 1 of SEQ ID NO: 1 (5'-AGAGGGATTCATGCTTCGGTTACCCACAGTCT) having a BamHI site and primer 2 (3'-GGAAAATTTTGCGGCCGCTTAGAACATGCCACCTCCCA) with SEQ ID NO: 2 having a NotI site. The total length of HSP60 cDNA (1722bp) was amplified by subcloning into the pcDNA3 (Invitrogen) expression vector. Human hsp60 cDNA cloned in pcDNA3 was truncated with BamHI and NotI and ligated to the pET28a vector (Novagen). Chimeric fusion proteins, consisting of 6X histidine linker proteins in addition to the total 573 amino acids of human HSP60, were expressed after induction with isopropyl β-D-thiogalactopyranoside in BL21 (DE3) bacteria. Fusion proteins were purified as inclusion bodies and used as immunogens for the production and purification of polyclonal antiserum in rats.

Immunization was performed by intraperitoneal administration of BALB / c mice with 200 μg of recombinant protein mixed with Freund's complete adjuvant, Gibco BRL Life Technologies, Inc., Grand Island, NY, USA. Two additional inoculations with 200 μg antigen, third inoculation with 200 μg antigen dissolved in the incomplete adjuvant, intervals were 3 weeks. A final infusion of 200 μg (without adjuvant) was administered 3 days prior to fusion and thereafter blood was drawn. Antiserum was obtained by centrifuging blood for 10,000 X g for 30 minutes. Splenocytes (2 × 10 ⁸ cells) were fused with SP2 / O mouse myeloma cells using 43% polyethylene glycol 50 (Sigma) to fuse splin cells with mouse myeloma. Cells were then plated in 96 wells, and containing hypo% containing 35% RPMI-1640 with 10% hybridoma enhancing medium (Sigma), 20% FBS (Hyclone), 35% SP2 / O and HEPES. It was grown in xanthine / aminopterin / thymidine medium. Two weeks later, the supernatants of the hybridomas were used to test the production of antibodies using Mono S-purified N-SMase ε according to the method of Ausubel.

Antibodies against human HSP60 were affinity-purified against the chimeric fusion proteins of 6 X His and HSP60.

Example 4. Cerebral Cortical Cells and Glia Cell Culture

Pure rat cerebral cortical neurons cultures were prepared from fetal Sprague-Dooli rats on day 16 of gestation. In summary, the isolated cells are balanced salt solution using a Pasteur pipette with no Ca ²⁺ and Mg ²⁺ Hanks: were isolated from (Hank's balanced salt solution, HBSS 1mM sodium pyruvate, 10mM HEPES pH 7.4). Isolated cells were plated in DMEM / F-12 in a poly-D-lysine coated culture plate. Growth of non-neuronal cells was inhibited by the addition of 10 μM β-D-cytosine arabinofuranoside (AraC) at 18 hours of plating. Cells were maintained at 95% atmosphere and 5% CO ₂ , 37 ° C.

Glia cell cultures were prepared from neocortis of postnatal rats (1 to 3 days) and per 6-well vessel with medium supplemented with 10% FBS and 10% horse serum. 0.25 to 0.5 hemispheres were plated. After two weeks in vitro, the cultures were changed weekly with the same medium for neuronal culture. Glia cell cultures were used to plate on days 14-28 in in vitro tests.

Example 5. [ ³ H] serine or [ ³ Lipid amount analysis using H] palmitate labeling

Cells were pre-labeled with [ ³ H] serine ( ³ μCi / ml) for 48 hours or with [ ³ H] palmitate (1 μCi / ml) for 24 hours. To measure the increase in ceramide by the de novo pathway, the cells were exposed to hypoxia in the presence of [ ³ H] serine or [ ³ H] palmitate, the precursor of ceramide. Cells were washed twice with PBS and exposed to hypoxia. Cold methanol: 0.5N-HCl (1: 1) solution was added and cells were scraped off the plate. Lipids were extracted according to the method of Bligh et al. And aliquots were counted for normalization. Lipids were incubated in 0.1N methanolic KOH for 1 hour at 37 ° C. to hydrolyze phospholipids. The resulting lipid was dried under nitrogen. Lipids were suspended in chloroform: methanol (1: 1, v / v) and spotted on TLC silica gel G plates.

The plate was developed in chloroform: methanol: water: acetic acid (85: 4.5: 5: 0.5, v / v / v / v) and dried under nitrogen again to give chloroform: methanol: water: acetic acid (65: 25: 8.8: 4.5, v / v / v / v) up to 50% of the total length. Under these conditions, sphingoid bases such as ceramide, glucosylceramide, sphinganine, sphingosine and SM are well separated. Commercially available lipid standards were chromatographed together. After drying, the lipids were visualized by iodine vapor staining. Spots were scraped off and mixed with 2.5 ml scintillation solution (Insta gel-XF, Packard instrument Co., Meriden, CT). Radioactivity was measured with a Packard tri-carbide β-scintillation counter.

Experimental Example 1. Immunoassay Analysis

1-1. Immunoprecipitation

 Antibodies to human HSP60 (200 mg) affinity-purified against the chimeric fusion proteins of 6 X His and HSP60 were coupled with 40 ml of protein G Agarose beads and the beads thus bound were It was blocked with 0.1 M Ethanolamine (pH 8.0) for 2 hours at room temperature and washed with 40 ml of PBS buffer. Protein G-agarose beads bound to the washed antibody were reacted with 20 mg of the active protein fraction obtained on an SP-5PW cationic exchange HPLC column. Reaction conditions were 20 mM Tris, pH 7.5, 10% glycerol, 0.1 mM EDTA, 100 mM NaCl, the reaction was centrifuged and the supernatant was taken to measure the N-SMase activity.

In response to parallel changes in the protein levels of the supernatant beads and supernatant (see FIG. 6), the antibody also significantly immunoprecipitated partially purified N-SMase activity in the bovine brain (see FIG. 7). ).

1-2. Immunoblotting analysis

In order to observe the presence of HSP60 in the beads and the possibility of a component in which the enzyme immunoprecipitates with HSP60, the protein in the precipitate was tested by silver staining and Western blotting analysis. While several spots with molecular weights of 62-, 60-, 58- and 57-kDa are seen in immunoprecipitates by silver staining (see FIG. 8), only 57-kDa spots are shown in immunoblotting assays. It was not detected (see FIG. 9). This suggests that the 57-kDa spot is a protein associated with HSP60.

Experimental Example 2 Measurement of SMase Activity in HSP60 Infected Cell Line

Human HSP60 cDNA was amplified with primer 1 of SEQ ID NO: 1 (5'-AGAGGGATTCATGCTTCGGTTACCCACAGTCT) with BamHI site and primer 2 of SEQ ID NO: 2 (3'-GGAAAATTTTGCGGCCGCTTAGAACATGCCACCTCCCA) with NotI site. The total length of HSP60 cDNA (1722bp) was amplified by subcloning into the pcDNA3 (Invitrogen) expression vector.

Human HEK 293 cells were maintained in humid 5% CO ₂ , 37 ° C. in DMEM medium supplemented with 10% FBS and 100 units / ml penicillin and streptomycin, and 2 mM glutamine. One day prior to infection, cells were dispensed into 6-well cell culture plates at a final density of 50-70% confluence (˜3 × 10 ⁵ cells / well). Cells were infected according to the manufacturer's suggestion using a mixture of Superfect (Qiagen) and pcDNA3 or pcDNA3 / HSP60.

Infected cells were incubated for 3-4 hours with the mixture, and then growth medium containing twice the normal serum concentration was added to the cells without removing the infection mixture. 48 hours after infection, cells were washed with PBS and homogenized by homogenization with homogenization buffer (25 mM Tris-HCl pH 7.0, 1 mM EDTA, 10 mM β-mercaptoethanol). Cell residue was removed by centrifugation at 1,000 X g for 10 minutes and then analyzed with lysate.

The activity of Western blot and SMase on HSP60 was measured in the infected cell lines.

Referring to the results of FIG. 10, it was confirmed that SMase activity was high in cells expressing HSP60 protein.

Experimental Example 3. Measurement of SMase Activity in Antisense HSP60 Expressing Cell Line

pcDNA3-HSP60 infected HEK 293 cells were infected with 4 μM of ODN (A-ODN or S-ODN) dissolved in DBS without FBS and antibiotics. After 3-4 hours of incubation with the mixture, growth medium containing twice the normal serum concentration was added to the cells without removing the infection mixture. 48 hours after infection, cells were washed with PBS and homogenized by homogenization with homogenization buffer (25 mM Tris-HCl pH 7.0, 1 mM EDTA, 10 mM β-mercaptoethanol). Cell residue was removed by centrifugation at 1,000 X g for 10 minutes and then analyzed with lysate.

Thiophosphate-modified A-ODN and control sense-ODN (S-ODN) specific for mammalian hsp60 (Steinhoff, et al . ; Proc. Natl. Acad. Sci . USA, 91 , pp5085) -5088, 1994). The A-ODN and S-ODN nucleotide sequences are shown in SEQ ID NOs: 3 and 4 (SEQ ID NO: 3 A-ODN, 5'-AGCATTTCTGCGGGG-3 '; SEQ ID NO: S-ODN, 5'-CCCCGCAGAAATGCT-3' ).

FIG. 11 is a diagram analyzing the expression and SMase activity of HSP60 protein in antisense A-ODN-treated cell lines. As the synthetic oligomers are completely complementary to their specific mRNAs, they inhibit N-SMase ε protein expression and enzyme activity. Revealed.

Experimental Example 4. siRNA Preparation and Infection

siRNAs suitable for hsp60 mRNA were designed with 2 base overhangs on 5 'phosphate, 3' hydroxyl groups and strands; It was chemically synthesized by Xeragon Co. The following gene-specific sequences have been used successfully: Si-HSP60 sense 5'-UGCUCACCGUAAGCCUUUGdTdT-3 'of SEQ ID NO: 5 and antisense 5'-dTdTACGAGUGGCAUUCGGAAAC-3' of SEQ ID NO: 6. Annealing for duplex siRNA formation was performed as described. Infection of siRNA to target hsp60 was performed with TransMessenger transfection reagent (Qaigen). siRNA (0.8 μg) was condensed with Enhancer R according to the manufacturer's recommendations and prepared with 4 μl of transmesin reagent. Infected complexes were diluted in 300 μl of cell growth medium (no serum or antibiotics included) and added directly to HEK 293 cells. After 4 hours, the cells were replaced with normal growth medium, and the cells were analyzed at 48 hours after infection. Cells were lysed and centrifuged. Hsp60 protein levels (18 μg) were confirmed on Western blot. SMase activity (110 μg) was determined using ¹⁴ C-sphingomyelin as substrate. All results are averaged from 3-4 independent experiments.

Referring to the results of Figure 12, comparing the activity of SMase between the treatment with hsp60 siRNA and nothing (control), it can be seen that the inhibition of protein synthesis by siRNA is less active than the control, Western blot It was confirmed that the amount of protein was reduced in parallel with the activity in the phase.

Experimental Example 5 Analysis of N-SMase ε Activity by TNF-α Treatment

In order to observe the change in activity according to the quantitative increase of N-SMase ε in the treatment of TNF-α in cerebral cortical neurons, the activity and the amount of ceramide, a hydrolysis product of N-SMase ε, were measured.

5-1. TNF-α treatment and N-SMase ε specific activity change test

Cerebral cortical neurons (three 6-well dishes / conditions) were washed once with PBS and incubated in DMEM medium supplemented with 1% FBS and 1 μCi / ml [ ³ H] serine. After 3 days, cells were washed three times with 10 ml PBS and then incubated for 1 day with DMEM containing 10% FBS. Before treatment with TNF-α, the amount of FBS was reduced to 2%, and after 2 hours, the cells were incubated with various concentrations of TNF-α, and then quickly transferred to culture at 37 ° C. The morphological changes of the cells were observed under the microscope, and a significant number of cells were killed by TNF-α 24 hours treatment compared to the control group (see FIG. 13).

In addition, in order to determine whether the death of cerebral cortical neurons by TNF-α is related to the change of N-SMase ε activity, the activity of N-SMase ε was measured at different time intervals after TNF-α treatment. .

Referring to the results of FIG. 14, it was confirmed that the N-SMase ε activity was increased by TNF-α in time-dependent manner, and at 30 minutes, the activity was increased by 2 times compared to the control group. Therefore, it was confirmed that the change of N-SMase ε activity is involved in neuronal cell death induced by TNF-α.

In addition, immunoblot analysis was performed using the N-SMase ε antibody prepared in Example 3 to determine the mechanism of increase of N-SMase ε activity by TNF-α. As a result, it was confirmed that the amount of N-SMase ε protein was increased compared to the control group when TNF-α was treated for 30 minutes in cerebral cortical neurons (see FIG. 15).

Therefore, it was confirmed that the N-SMase ε activity increased after TNF-α treatment in cerebral cortical neurons due to quantitative increase.

5-2. Ceramide Test Analysis Generated by N-SMase ε Activity

Activity of N-SMase ε is increased by TNF-α After confirming through the above experiments, the quantitative change of ceramide, a hydrolyzate of N-SMase ε, was observed in this experiment.

3 μM of Fumonisin B1 was added 10 minutes before TNF-α treatment. The resulting ceramide was isolated on TLC as follows; Lipids were first separated using chloroform: methanol: acetic acid: water (170: 9: 10: 1; v / v / v / v), then the plates were dried and chloroform / methanol / acetic acid / water as secondary solvent (39 : 15: 5.3: 2.7; v / v / v / v). Individual lipids were visualized by iodine vapor staining. Radioactive spots corresponding to SM and ceramide were scraped off and radioactivity was measured by β-liquid scintillation counter.

In the results of FIG. 16, the amount of ceramide was increased according to the treatment of TNF-α in the cerebral cortical neurons at different times, and at the same time, the amount of sphingomyelin which is a substrate of N-SMase ε was decreased. . In addition, after pretreatment with FB1, a biosynthetic ceramide production inhibitor, quantitative change of ceramide by TNF-α was confirmed to have no effect. Therefore, it could be confirmed that the increase in ceramide amount by TNF-α was not due to an increase in ceramide biosynthesis but rather an increase in N-SMase ε activity.

Neutral SMase ε and its antibodies and antisenses of the present invention are used to modulate the production of ceramides, which are important signals for various apoptosis or inflammatory reactions, and thus act as anti-cancer or anti-inflammatory agents by acting as an inhibitor of apoptosis and inhibiting inflammation. It can be used as a medicine.

<110> KIM, Dae-Kyong <120> Novel sphingomyelinase, the antibody against it, the antisense and the          preparation method <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> HSP60_BamHI primer 1 <400> 1 agagggattc atgcttcggt tacccacagt ct 32 <210> 2 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> HSP60_NotI primer2 <400> 2 ggaaaatttt gcggccgctt agaacatgcc acctccca 38 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> A-ODNs <400> 3 agcatttctg cgggg 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> S-ODNs <400> 4 ccccgcagaa atgct 15 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> siRNA primer 1 <220> <223> Si RNA HSP60 <400> 5 ugcucaccgu aagccuuugd tdt 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> siRNA primer2 <220> <223> siRNA HSP60 primer2 <400> 6 dtdtacgagu ggcauucgga aac 23

Claims (7)

Bovine brain was mixed with homogenization buffer to homogenize and centrifuged to remove cell debris and nuclei, and then centrifuged to resuspend and stir the pellets in buffer containing ammonium sulfate, followed by centrifugation. A first step of obtaining an enzyme source 'ammonium sulfate extract' for purifying the pingomyelinase (N-SMase) ε; A second step of passing the first ammonium sulfate extract through a DEAE-cellulose column and eluting with a buffer containing ammonium sulfate and Triton X-100 to obtain an active fraction; A third step of passing the active fraction of step 2 through a butyl-toyopearl column and eluting with tertiary distilled water to obtain an active fraction; A fourth step of passing the three active fractions through DEAE-5PW HPLC and eluting with a buffer containing ammonium sulfate and Triton X-100 to obtain an active fraction; A fifth step of passing the active fraction of step 4 through phenyl-5PW HPLC and eluting with tertiary distilled water to obtain an active fraction; The 58,000 active purified fractions were passed through a mono S cation exchange HPLC and eluted with a buffer containing sodium chloride and Triton X-100 to obtain 38000-fold purified membrane-associated 60 kDa. Method for isolation and preparation of neutral sphingomyelinase (N-SMase) ε. delete delete delete delete delete delete

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