JP3705763B2 - Cholesterol measurement probe and cholesterol measurement method - Google Patents

Description

【図面の簡単な説明】
【図１】野生型コレステロール酸化酵素（pCO117）とコレステロール酸化酵素変異体（V145）のSDS-PAGEおよびウエスタンブロッティング分析の結果である。
【図２】巨大リポソームに含まれるコレステロールにコレステロール酸化酵素変異体が結合することを示した顕微鏡写真である。
【図３】高張および低張条件下で、それぞれmethyl-β-cyclodextrinで処理した場合と処理しない場合の免疫染色の結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cholesterol measuring method capable of easily and accurately detecting cholesterol contained in various biological samples and foods and a material therefor.
[0002]
[Prior art]
Cholesterol is a typical animal metabolite. It is a raw material for the biosynthesis of adrenocortical hormones, sex hormones and bile acids, and is a constituent of biological membranes such as cell membranes and plasma lipoproteins.
[0003]
Cholesterol in the human body is mostly synthesized in the body (about 1.5 to 2.0 g), but about 0.3 g is also supplied from food. Cholesterol in food is digested and absorbed along with other fats and taken up into kilomicrons, but most of the absorbed cholesterol (80-90%) is esterified with long chain fatty acids, and this cholesterol ester is often It adheres to the inner wall of blood vessels together with fat and causes severe diseases such as arteriosclerosis.
[0004]
Therefore, accurately grasping the amount of cholesterol in living organisms (for example, biological membranes and blood) or cholesterol contained in foods is important for the prevention of various diseases caused by excess cholesterol and the determination of strategies for treatment. is there.
[0005]
For this reason, various methods for measuring cholesterol and reagents therefor have been developed. In particular, JP-A-10-080300, JP-A-10-311833, JP-A-2001-231597, and the like are known as measuring reagents using cholesterol oxidase having excellent binding activity to cholesterol. In addition, a mutant of cholesterol oxidase (Japanese Patent Laid-Open No. 08-242860) and a method for measuring cholesterol using this enzyme mutant (Japanese Patent Laid-Open No. 2000-224999) are also known.
[0006]
[Problems to be solved by the invention]
As described above, many attempts have been made to use cholesterol oxidase for cholesterol measurement. Cholesterol measurement using such cholesterol oxidase and its variants measures cholesterol by detecting changes such as degradation and modification of the substrate (cholesterol) by cholesterol oxidase as physical signals. is there.
[0007]
On the other hand, as a method for measuring proteins and the like simply and with high accuracy, there is a method for detecting the presence or absence of a target protein or quantifying its concentration using a ligand having a high affinity for the protein or the like as a probe. Although widespread, no such probe is known for cholesterol.
[0008]
The present invention has been made in view of the circumstances as described above, and has a probe having high affinity for cholesterol and a new measurement for easily and accurately measuring cholesterol using this probe. The challenge is to provide a method.
[0009]
[Means for Solving the Problems]
As an invention for solving the above-mentioned problems, this application is a cholesterol measurement probe comprising a cholesterol oxidase mutant having cholesterol binding activity but low oxidation / isomerization reaction activity for cholesterol, and a reporter substance I will provide a.
[0010]
In the measurement probe of the present invention, the cholesterol oxidase mutant has a deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1, or one or more amino acid residues substituted with other amino acid residues. It has a preferred embodiment that it has an amino acid sequence, more specifically, a cholesterol oxidase variant has an amino acid sequence in which the 145th valine residue in the amino acid sequence of SEQ ID NO: 1 is substituted with a glutamine residue.
[0011]
Furthermore, another preferable aspect is that the cholesterol oxidase mutant is an N-terminal acetylated, glycosylated, myristoylated, isoprenylated, or phosphorylated cholesterol oxidase.
[0012]
Furthermore, in this cholesterol measurement probe, another embodiment is that the reporter substance is an enzyme, a fluorescent protein or a fluorescent substance.
The invention of this application also comprises a method for measuring cholesterol, comprising: reacting any one of the cholesterol measurement probes with a test substance, and measuring a change amount of a detection signal emitted from a reporter substance bound to the measurement probe; Each of the cholesterol measurement kits including any of the cholesterol measurement probes as an essential component is provided.
[0013]
Hereinafter, embodiments of these inventions will be described in detail.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
In the invention, the measurement of cholesterol means detection of the presence or absence of cholesterol and quantification of the amount (concentration) of cholesterol. The cholesterol to be measured includes cholesterol derivatives such as cholesterol ester, LDL-cholesterol, and HDL-cholesterol in addition to cholesterol itself.
[0015]
The cholesterol measuring probe of the present invention is characterized by comprising a cholesterol oxidase mutant and a reporter substance.
The cholesterol oxidase mutant is an enzyme mutant that is artificially modified so that its cholesterol binding activity is similar to that of the wild-type enzyme, but its oxidation / isomerization reaction activity (catalytic activity) to cholesterol is reduced. Such mutants are, for example, deletion or addition of one or more amino acid residues in the amino acid sequence of wild-type cholesterol oxidase (SEQ ID NO: 1), or one or more amino acid residues as other amino acid residues. Those having a substituted amino acid sequence can be used. Such a cholesterol oxidase mutant is obtained by introducing a mutation into a polynucleotide encoding cholesterol oxidase (for example, DNA registered as GenBank Accession No. M31939) and expressing it by an appropriate host-vector system. It is obtained by evaluating the binding activity and catalytic activity of the obtained mutant enzyme with cholesterol, repeating this as necessary, and selecting a more preferable mutant enzyme (ie, having high cholesterol binding activity and low catalytic activity). .
[0016]
Many methods are known for introducing mutations into polynucleotides. For example, the classical method using a transposon or a mutagen (Science, 229: 1193-1201, 1985), Error-prone PCR (Technique, 1: 11-15, 1985), a method of replacing specific regions with random sequences by random chemical synthesis of DNA (Nature Struct. Biol. 3: 446-451, 1996; Proc. Natl. Acad. Sci. USA. 93: 5590-5594, 1996) and sexual PCR method (Nature 370: 389-391, 1994).
[0017]
This invention provides a cholesterol oxidase variant in which the 145th valine residue of the amino acid sequence of SEQ ID NO: 1 is substituted with a glutamine residue as a particularly preferred embodiment.
[0018]
The cholesterol oxidase mutants also include cholesterol oxidase that has undergone various modifications such as N-terminal acetylation, glycosylation, myristoylation, isoprenylation, or phosphorylation. Such a mutant can be prepared by a known method according to each modification. Further, for example, when a DNA sequence encoding cholesterol oxidase is expressed in a host-vector system, it can also be obtained by post-translational protein modification in the host cell.
[0019]
Specific examples of the reporter substance that binds and integrates with the cholesterol measurement probe of the present invention include enzymes, fluorescent proteins, fluorescent substances, and the like. More specifically, the enzyme is not particularly limited as long as it has an ability to specifically bind to a substrate and catalyze a reaction, for example, protease, nuclease, alkaline phosphatase, β-galactosidase, Examples include luciferase, glucose oxidase, chloramphenicol acetyltransferase, and the like. Changes such as degradation and modification of the substrate due to these enzyme activities can be detected as physical signals.
[0020]
Examples of the fluorescent protein include green fluorescent protein (GFP) and its mutant (EGFP). Examples of the exogenous fluorescent substance include fluorescent dyes such as fluorescein series, rhodamine series, eosin series, NBD (7-nitrobenz-2-oxa-1,3-diazole) series. Since these fluorescence changes sensitively according to the surrounding environment, the change can be detected by a physical method.
[0021]
The cholesterol measuring probe of the present invention may be one in which the cholesterol oxidase mutant and the reporter substance are combined and integrated. When the above-described labeling enzyme or fluorescent protein is used as a reporter substance, for example, a polynucleotide encoding each protein is linked and expressed as a mutant enzyme-labeling enzyme fusion protein or a mutant enzyme-fluorescent protein fusion protein. What should I do? When a fluorescent dye is used as a reporter substance, for example, a method through non-covalent bonding such as modification of hemoglobin heme with a fluorescent dye, or a cysteine residue of a mutant enzyme is chemically modified with a fluorescent dye. The two can be combined and integrated by a method such as through a covalent bond. Alternatively, the cholesterol oxidase mutant and the reporter substance may be separate and may be integrated at the time of cholesterol measurement. For example, a ligand (for example, an antibody) that specifically binds to a cholesterol oxidase variant may be labeled with a reporter substance, and the cholesterol oxidase variant and the labeled antibody may be mixed when measuring cholesterol.
[0022]
The cholesterol measurement method of the present invention detects and quantifies cholesterol contained in a test substance by reacting the cholesterol measurement probe with the test substance and measuring the amount of change in the detection signal emitted from the reporter substance of the measurement probe. It is characterized by doing.
[0023]
Examples of test substances include various biological samples (cells, blood), various foods, and the like. The cholesterol to be measured includes cholesterol ester, LDL-cholesterol, HDL-cholesterol and the like in addition to cholesterol itself.
[0024]
One embodiment of this measuring method is a detection signal generated from a reporter substance labeled with this antibody by reacting the above-mentioned cholesterol oxidase mutant with a test substance, further binding a ligand to the cholesterol oxidase mutant. A change in (fluorescence intensity or enzyme activity) is measured by a known means. The ligand for the cholesterol oxidase mutant is, for example, a polyclonal antibody or a monoclonal antibody prepared by a known method using wild-type cholesterol oxidase or a mutant thereof as an antigen.
[0025]
In another embodiment of this measurement method, a cholesterol measurement probe in which a cholesterol oxidase mutant and a reporter substance are integrated is reacted with a test substance, and a change in a signal generated by the reporter substance of this probe is measured by a known means.
[0026]
In this measurement method, “change in detection signal” includes not only increase / decrease in signal “intensity” but also change in “property” of signal. Specifically, in addition to the increase / decrease in absorbance, fluorescence intensity, etc., there is a shift in fluorescence wavelength.
[0027]
The cholesterol measurement kit of the present invention comprises the above-described cholesterol measurement probe as an essential component, and can be used in the above cholesterol measurement method. Such a measurement kit is provided with the same configuration as a measurement kit using a reaction between a normal receptor protein and a ligand. That is, the measurement kit of the present invention includes at least the above-described cholesterol measurement probe, and may further include a diluent, a washing solution, and positive control cholesterol as optional elements.
[0028]
When detecting the presence or absence of cholesterol using this measurement kit, for example, when comparing the signal intensity of the reporter substance with or without adding a probe to the test substance, or using a positive control if necessary And the signal intensity of the reporter substance. In addition, when quantifying the cholesterol concentration, for example, a calibration curve may be prepared using an appropriate sample and quantified based on this.
[0029]
Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited by the following examples.
[0030]
【Example】
1. Materials and methods
1.1. Mutant cholesterol oxidase and its antibody
Cholesterol oxidase gene derived from Streptomyces sp. SA-COO (J. Bacteriol. 171 (1): 596-601, 1989) is described in the literature (Protein Eng. 11 (11): 1075-1081, 1998). Mutation was carried out in the same manner to prepare mutant enzyme V145Q in which the 145th valine residue in the amino acid sequence of cholesterol oxidase (SEQ ID NO: 1) was replaced with a glutamine residue. Expression and purification of wild-type and V145Q enzymes were carried out according to the method described in the literature (Protein Eng. 11 (11): 1075-1081, 1998), but only Escherichia coli culture was carried out at the final concentration under conditions of 18 ° C for 15 hours. Expression and purification were carried out after induction with 10 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Enzyme purity was evaluated by SDS-polyacrylamide gel electrophoresis on a gel containing 10% (w / v) acrylamide by the method of Laemmli (Nature 227: 680-685, 1970), and Coomassie Brilliant Blue R-250 was used. Stained. Anti-cholesterol oxidase polyclonal antibody was produced by immunizing male rabbits with 200 mg of purified wild-type enzyme emulsified in Freund's complete adjuvant (Calbiochem). Two weeks later, the rabbits were boosted with 200 mg of purified cholesterol oxidase emulsified in Freund's incomplete adjuvant (Calbiochem). Two weeks later, blood was collected from this rabbit, and serum was obtained by clotting at 37 ° C. for 5 hours. Standard Western blotting was performed to confirm the immune response of the anti-cholesterol oxidase antibody to this enzyme (Antibodies: A laboratory manual. Cold Spring Harbor laboratories, Cold Spring Harbor, New York, 1988).
1.2. Preparation of liposomes The preparation of giant liposomes was basically as described in the literature (Biophys. J. 64: 676-685, 1993; Biophys. J. 71: 3242-3250, 1996). That is, phosphatidylcholine and phosphatidylserine were mixed in chloroform to a weight ratio of 19: 1 to prepare a 10 mg / ml control lipid solution. Phosphatidylcholine, phosphatidylserine and cholesterol were mixed in chloroform to a weight ratio of 15: 1: 4 to prepare a 10 mg / ml cholesterol-containing lipid solution. 100 ml of the lipid mixture placed in a glass test tube was dried with a stream of N ₂ gas to remove chloroform. 4 ml of 0.1M sucrose was gently added to the thin lipid layer formed at the bottom of the test tube at room temperature. After incubation at 37 ° C. for 2 hours, this solution containing giant liposomes was diluted with 0.1 M glucose and applied to the reaction with the mutant enzyme V145Q labeled with fluorescein nisothiocyanate (FITC).
1.3. Immunostaining hypertonic conditions: ECV304 cells grown in DMEM containing 10% fetal calf serum and 1% penicillin / streptomycin were incubated at 37 ° C. for 5 minutes under hypertonic conditions at a final concentration of 0.45 M sucrose. Purified cholesterol oxidase was added to this medium at 37 ° C. for 5 minutes so that the final concentration was 10 ng / ml. After washing with phosphate buffered saline (PBS), the cells on the cover glass were fixed with 4% paraformaldehyde at room temperature for 1 hour, and washed 3 times with PBS (10 minutes each). Then, it was treated with 0.1 M glycine for 15 minutes. Cells were washed twice with PBS (5 minutes each) and blocked with 5% skim milk dissolved in PBS.
Normal conditions: ECV304 cells were grown in DMEM containing 10% fetal calf serum and 1% penicillin / streptomycin, fixed with 4% paraformaldehyde for 1 hour at room temperature, and washed 3 times with PBS (10 minutes each) ), And treated with 0.1 M glycine for 15 minutes. Cells on the cover glass were washed twice with PBS (5 minutes each), purified cholesterol oxidase was added to a final concentration of 30 ng / ml, and incubated at 37 ° C. for 60 minutes. After rinsing with PBS, cells were fixed with chilled methanol for 3 minutes at -20 ° C, washed 3 times with PBS (5 minutes each), and blocked with 5% skim milk dissolved in PBS .
[0031]
Cells are reacted with anti-cholesterol oxidase antibody for 15 hours at 4 ° C, washed 3 times with PBS (10 minutes each), and added with rhodamine-conjugated secondary antibody (Cappel) for 3 hours at room temperature. Incubated with After 3 washes with PBS (5 minutes each), the samples were embedded using PermaFluor (Themo Shandon). Fluorescence images were taken using a cooled CCD camera (MetaView) on an Axiophot microscope (Carl Zeiss) or an LMS410 confocal laser scanning microscope (Carl Zeiss).
2.Result
2.1. Liposomes for measuring cholesterol in cholesterol Variant cholesterol oxidase bound specifically to cholesterol inside giant liposomes. In this example, a cholesterol oxidase mutant having an amino acid substitution (V145Q) was used. The reason for selecting this mutant V145Q is that the level of oxidative-isomerization activity is low (1/4) compared to the wild-type enzyme and nevertheless retains normal binding activity to cholesterol. For this reason, V145Q does not dissociate as soon as it binds to cholesterol. The wild-type and V145Q enzymes purified by affinity column chromatography contain a single band with a molecular weight of approximately 55 kDa in sodium dodecyl sulfate (SDS) polyacrylamide gel, which uses an antibody prepared against the wild-type enzyme. Were specifically detected (FIG. 1). To examine the ability of purified V145Q to bind to cholesterol on membranes, unilamellar liposomes were prepared and incubated with FITC-labeled V145Q. As shown in FIG. 2, the mutant enzyme binds to the cholesterol-containing giant liposome, but does not bind to the cholesterol-free liposome. From these results, it was confirmed that cholesterol on unilamellar liposomes can be specifically detected using V145Q.
2.2. Cholesterol measurement of cell membrane Cholesterol oxidase mutants stably bind to cholesterol on the plasma membrane, but membrane cholesterol is rapidly internalized in intracellular organelles, so that the detection signal increases due to cellular uptake. there's a possibility that. Therefore, it is necessary to suppress uptake by endocytosis in order to specifically detect only cholesterol on the plasma membrane. Therefore, ECV304 cells were incubated in a hypertonic solvent and reacted with a cholesterol oxidase mutant in a state in which uptake into cells was suppressed. As a result, it was confirmed that the mutant enzyme V145 stably binds to cholesterol in the plasma membrane (FIG. 3).
[0032]
In addition, methyl-β-cyclodextrin, a cholesterol-specific reagent, removes cholesterol from the plasma membrane (J. Biol. Chem. 270: 17250-17256, 1995; J. Biol. Chem. 271: 16026-16034). 1996), when cholesterol was removed from the cell membrane using this methyl-b-cyclodextrin, the signal detected by the combination of the mutant enzyme V145Q and its antibody was attenuated (FIG. 3). These results also confirmed that V145Q specifically binds to cholesterol on the plasma membrane.
[0033]
【The invention's effect】
As explained in detail above, the invention of this application makes it possible to easily and accurately measure cholesterol contained in biological samples and foods. The present invention is useful for preventing or diagnosing diseases caused by cholesterol, determining a therapeutic strategy, and the like.
[0034]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the results of SDS-PAGE and Western blotting analysis of wild-type cholesterol oxidase (pCO117) and cholesterol oxidase mutant (V145).
FIG. 2 is a photomicrograph showing that a cholesterol oxidase mutant binds to cholesterol contained in giant liposomes.
FIG. 3 shows the results of immunostaining with and without treatment with methyl-β-cyclodextrin under hypertonic and hypotonic conditions, respectively.

Claims (7)

A cholesterol measurement probe comprising a cholesterol oxidase mutant having cholesterol binding activity but low activity in oxidizing and isomerizing cholesterol, and a reporter substance. The cholesterol oxidase variant has a deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence in which one or more amino acid residues are substituted with other amino acid residues. Cholesterol measurement probe. The cholesterol measurement probe according to claim 2, wherein the cholesterol oxidase mutant has an amino acid sequence in which the 145th valine residue in the amino acid sequence of SEQ ID NO: 1 is substituted with a glutamine residue. The cholesterol measuring probe according to claim 1, wherein the cholesterol oxidase variant is N-terminal acetylated, glycosylated, myristoylated, isoprenylated, or phosphorylated cholesterol oxidase. 5. The cholesterol measurement probe according to claim 1, wherein the reporter substance is an enzyme, a fluorescent protein, or a fluorescent substance. 6. A method for measuring cholesterol, comprising reacting the cholesterol measurement probe according to any one of claims 1 to 5 with a test substance, and measuring a change amount of a detection signal emitted from a reporter substance bound to the measurement probe. A cholesterol measurement kit comprising the cholesterol measurement probe according to claim 1 as an essential component.

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