JP5773405B2 - Method for detecting compounds that interact with molecules on cell membranes - Google Patents

Description

  The present invention relates to a method for detecting a compound that interacts with a molecule on a cell membrane, and a kit for carrying out the method.

  In the phospholipid bilayer constituting the cell membrane, there are many proteins, lipids other than phospholipid, and the like, and some function as growth factor receptors, cell adhesion factors, ion channels, and the like. These molecules on the cell membrane can move relatively freely in the cell membrane, and are repeatedly assembled and dissociated. In particular, what is called raft, which is a collection of multiple molecules on the cell membrane and exposed on the cell membrane surface, is an important role such as a receptor for bacteria and viruses and a platform for extracellular information to be transmitted into the cell. Take on. Therefore, it is extremely important in biochemical studies to know whether a molecule on a cell membrane functions in cooperation with any other molecule on the cell membrane.

  However, analysis of interactions between molecules on cell membranes is very difficult. For example, as a method for separating and detecting a protein that interacts with a certain target protein, an immunoprecipitation method in which an antibody specific to the target protein is allowed to act to cause selective precipitation is known. However, with this method, it is difficult to reflect the state in which the target protein and other proteins interact under physiological conditions. This is because, in this method, even when other proteins interact with the target protein on the cell membrane, the interaction may be eliminated when the target protein is separated from the cell membrane. . In addition, a pseudo-interaction may occur at the protein separation stage even though it does not interact on the cell membrane of a living cell.

  In addition, there is known a cross-linker method in which a cross-linker is bound to a target protein and specified by cross-linking with an interacting protein. However, in this method, since the length and shape of the crosslinker molecule are fixed, only a part of the tightly bound protein is detected by crosslinking. As a result, there are few successful examples in analyzing the interaction between molecules on the cell membrane by the crosslinker method.

  Therefore, a technique specialized in detecting an interaction between molecules on a cell membrane by a method other than the above has been studied.

  The present inventors have developed a technique for detecting an interaction between molecules on a cell membrane (Patent Document 1). In this technique, first, a compound (first reagent) that selectively binds to a target molecule on the cell membrane and promotes radicalization, such as horseradish peroxidase (HRP), is allowed to act on the cell. A compound having a group to be converted and a labeling group (second reagent) is allowed to act. As a result, the second reagent is radicalized by HRP or the like of the first reagent selectively bound to the target molecule, and binds to a compound existing in the vicinity of the target molecule. Since the second reagent has a labeling group, the compound that interacted with the target molecule in the cell membrane can be identified. Patent Document 1 exemplifies biotin, a fluorescent coloring group, a radioisotope-containing group, a labeled peptide, and a hapten as the labeling group of the second reagent.

International Publication No. 2008/093595 Pamphlet

  As described above, the present inventors have developed a technique for detecting a compound that interacts with a target molecule in a cell membrane (Patent Document 1). However, there was still a problem with this technology.

  That is, when the reaction was carried out using the reagent specifically disclosed in Patent Document 1, many compounds were labeled even when the first reagent that selectively binds to the target molecule was not used. It was sometimes difficult to detect compounds that interact with molecules.

  Accordingly, the present invention aims to provide a method capable of highly accurately detecting a compound that interacts with a target molecule in a cell membrane by eliminating non-selective binding of a labeling compound as much as possible, and a kit that can be used in the method. And

  The inventors of the present invention have made extensive studies to solve the above problems. As a result, while the above Patent Document 1 exemplifies many substituents as a labeling group, the only reagents actually synthesized were those having biotin as a labeling group. It was found that non-selective binding can be effectively eliminated by using a reagent having a derivative group, and a compound that interacts with a target molecule can be detected with high accuracy, and the present invention has been completed.

  Specifically, the reagent of Patent Document 1 having biotin as a labeling group sometimes cannot eliminate non-selective binding. That is, even when the first reagent is used or not, many compounds that do not interact with the target molecule may be labeled with the second reagent. According to the experiments by the present inventors, such non-selective binding was particularly seen in a fraction containing relatively heavy intracellular organelles such as the nucleus, mitochondria, and peroxisome. The cause was thought to be taken up into cells and radicalized by intracellular peroxidase.

  On the other hand, according to the method of the present invention, the non-selective binding as described above was remarkably suppressed, and a compound interacting with the target molecule could be detected with higher accuracy. The reason is that, initially, it was predicted that the second reagent could not pass through the cell membrane because the fluorescein derivative group was structurally larger than the biotin group. It was revealed that the two reagents were also taken up into the cells in a temperature-dependent manner. Further, when a second reagent having a 4-fluoro-7-nitrobenzofurazane group that is a fluorescent chromophore group as in the fluorescein group was used, non-selective binding could not be sufficiently suppressed. According to the method of the present invention, although the reason why the non-selective binding is remarkably suppressed despite the incorporation of the second reagent into the cell is not necessarily clear, the selection of the labeling group shows a marked improvement in accuracy. It was amazing.

A method for detecting a compound that interacts with a molecule on a cell membrane according to the present invention comprises:
Allowing a compound having a selective binding moiety to a molecule on a target cell membrane and a radicalization-promoting moiety to act on a cell;
Further allowing the compound represented by formula (I) to act on cells;
XYZN ₃ (I)
[Wherein, X represents a fluorescent fluorescein derivative group, Y represents a linker group, and Z represents an optionally substituted C _6-12 arylene group]
And a step of identifying a compound to which compound (I) is bound.

In the present invention, the “C _6-12 arylene group” means a divalent aromatic hydrocarbon group having 6 to 12 carbon atoms. Examples thereof include a phenylene group, an indenylene group, a naphthylene group, a biphenylene group, and the like, and preferably a phenylene group.

The substituent of the C _6-12 arylene group is not particularly limited as long as it does not inhibit the reaction. For example, a C _1-6 alkyl group, a C _1-6 alkoxy group, a C _1-7 acyl group, a hydroxyl group, a carboxy group And one or more substituents selected from the group consisting of a halogen atom, a nitro group, and a cyano group.

In the present invention, the “C _1-6 alkyl group” means a linear or branched monovalent aliphatic hydrocarbon group having 1 to 6 carbon atoms. Examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group, a pentyl group, an isoamyl group, and a hexyl group, and a C _1-4 alkyl group. Is more preferable, a C _1-2 alkyl group is more preferable, and a methyl group is more preferable.

The “C _1-6 alkoxy group” means a linear or branched aliphatic hydrocarbon oxy group having 1 to 6 carbon atoms. For example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, t-butoxy group, n-pentoxy group, n-hexoxy group, etc., preferably C _1-4 alkoxy Group, more preferably a C _1-2 alkoxy group, and most preferably a methoxy group.

The “C _1-7 acyl group” means a formyl group or a carbonyl group substituted with the above C _1-6 alkyl group. For example, a formyl group, an acetyl group, an ethylcarbonyl group, an isopropylcarbonyl group, a t-butylcarbonyl group, an isoamylcarbonyl group, an n-hexylcarbonyl group, and the like can be given. A C _2-4 acyl group is preferable, and a C _{2− 3 An} acyl group is more preferable, and an acetyl group is more preferable.

  Examples of the “halogen atom” include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a chlorine atom or a bromine atom, and more preferably a chlorine atom.

The number of substituents on the C _6-12 arylene group may be one or more as long as substitution is possible. Moreover, when there are a plurality of the substituents, the substituents may be the same as or different from each other.

  In the method of the present invention, the radicalization promoting moiety can include horseradish peroxidase or a heme compound, and the fluorescent fluorescein derivative group can include a fluorescein group.

  Moreover, SDS-PAGE can be mentioned as a specific method of the compound which compound (I) couple | bonded.

The kit according to the present invention is a kit for carrying out the above-described method of the present invention,
A compound having a selective binding moiety to a molecule on a target cell membrane and a radicalization-promoting moiety, and a compound represented by the formula (I) XYZN ₃ (I)
[Wherein, X represents a fluorescent fluorescein derivative group, Y represents a linker group, and Z represents an optionally substituted C _6-12 arylene group]
It is characterized by including.

  According to the method of the present invention, a compound that interacts with a biomolecule present on a cell membrane can be widely detected with extremely high accuracy using living cells. In doing so, it is not necessary to predict in advance, isolate and use compounds that will interact. Moreover, the method according to the invention can be carried out very simply and at low cost. Furthermore, according to the method of the present invention, it is possible to directly detect the above-mentioned compound by a normal fluorescent compound detection means without using a secondary antibody or the like. Therefore, the method of the present invention and the kit for carrying out the method of the present invention are extremely important industrially as being usable not only for biochemical research but also for drug development.

FIG. 1 is a photograph of an SDS-PAGE gel showing the result of carrying out the method according to the present invention (EMARS (Enzyme-Mediated Activation of Radical Source) reaction). FIG. 2 is a photomicrograph showing the distribution of the second reagent according to the present invention. A shows the results with human cervical cancer cells, and B shows the results with human B-cell lymphoma cells. FIG. 3 is a photograph of an SDS-PAGE gel showing the results of carrying out the method according to the present invention (EMARS reaction). FIG. 4 is a photograph of an SDS-PAGE gel showing the results of carrying out the method according to the present invention (EMARS reaction). FIG. 5 is a photograph of an SDS-PAGE gel showing the results of carrying out the method according to the present invention (EMARS reaction). FIG. 6 is a photograph of an SDS-PAGE gel showing the results of performing an EMARS reaction using a second reagent having NBD. FIG. 7 is a photograph of an SDS-PAGE gel showing the results of performing an EMARS reaction using a second reagent having a biotin group as a labeling group. FIG. 8 is a photograph of a gel showing the results of separation by SDS-PAGE of bovine serum albumin (BSA) joined with the second reagent according to the present invention. FIG. 9 is a photograph of an SDS-PAGE gel showing the result of purifying and concentrating the EMARS reaction product from the sample subjected to the method according to the present invention (EMARS reaction) using immunoprecipitation.

A method for detecting a compound that interacts with a molecule on a cell membrane according to the present invention comprises:
A step of causing a compound having a selective binding moiety to a molecule on a target cell membrane and a radicalization-promoting moiety to act on a cell (hereinafter referred to as “first step”);
A step of further causing the compound represented by formula (I) to act on cells (hereinafter referred to as “second step”);
XYZN ₃ (I)
[Wherein, X represents a fluorescent fluorescein derivative group, Y represents a linker group, and Z represents an optionally substituted C _6-12 arylene group]
A step of identifying a compound to which compound (I) is bound (hereinafter referred to as “third step”)
including.

  The method of the present invention detects a compound that interacts with a biomolecule present on a cell membrane. Therefore, according to the method of the present invention, it becomes possible to perform basic research on the role of a target molecule on a cell membrane, or to detect a difference in interaction between tissues. For example, the difference between cancer cells and normal cells can be clarified, and information for developing therapeutic agents may be provided.

  There are various molecules on the cell membrane. For example, the protein present on the cell membrane includes a surface protein present in the surface layer of the phospholipid bilayer and an endogenous protein at least partially present in the phospholipid bilayer. In the present invention, “existing on the cell membrane” means that at least a part is exposed to the outside of the cell membrane. For example, as protein on the cell membrane, a part of the protein is present inside the cell and a part of the protein is exposed to the outside of the cell membrane, a protein directly bound to phospholipid, or a cell membrane via lipid or oligosaccharide And those that are further bound to proteins exposed outside the cell membrane. The function of the protein is not particularly limited, and for example, functions such as transmitting information from a compound derived from the outside of the cell to the inside of the cell, or transmitting information inside the cell to the outside of the cell, and the like are considered.

  The type of molecule on the cell membrane is not particularly limited. For example, in addition to the proteins described above, there are lipids. In addition, some or all of the sugar chains bound to the protein may be exposed outside the cell membrane, and such sugar chains are also included. In particular, a lipid containing a sugar chain is one of the important components of a cell membrane, the lipid part is present in the phospholipid bilayer, and the sugar chain part is exposed outside the cell. This glycolipid is present in a concentrated manner in the raft and is considered to be involved in the generation of the raft.

  The type of “interaction” is not particularly limited, and refers to not only a specific bond such as a covalent bond or an electrostatic bond but also a case where a protein and one or more compounds are relatively close to each other and exhibit a function.

  The compound that interacts with the target molecule on the cell membrane is not particularly limited. For example, proteins, glycolipids, phospholipids, cholesterol, etc. that exist in the cell membrane, and information transmitters derived from the outside such as ions and ligands Can be illustrated.

  Hereinafter, the method of the present invention will be described in detail in the order of execution.

(1) First Step In the present invention, first, a compound having a selective binding moiety to a target cell membrane molecule and a radicalization promoting moiety (hereinafter sometimes referred to as “first reagent”) is added to a cell. Make it work. By this step, a moiety having a radicalization function can be selectively bound to a target molecule on the cell membrane.

  What is necessary is just to select suitably the molecule | numerator on the cell membrane used as the target which should identify the compound which interacts with the molecule | numerator, and there is no restriction | limiting in particular in the kind etc. The selective binding moiety to the cell membrane molecule can be appropriately selected depending on the target cell membrane molecule. For example, an antibody specific for a molecule on a cell membrane, a peptide that specifically binds to a sugar chain bound to the molecule, and the like can be mentioned. Preferably, an antibody is used. In addition, the selective binding moiety may be an antibody that secondarily labels the primary antibody when the primary antibody is bound to a molecule on the target cell membrane. That is, the first reagent used in the present invention may be a combination of a primary antibody against a target cell membrane molecule and a secondary antibody having a portion that binds to the primary antibody and has a radicalization function.

  The radicalization promoting moiety refers to a moiety that can radicalize the azide group of the compound (I) used in the second step described later. When radicalizing a compound, a peroxide such as hydrogen peroxide or light is usually used, which can damage live cells. Since the method of the present invention is characterized in that an interaction between compounds in the cell membrane of a living cell can be detected, those that damage a living cell are not used. Examples of such radicalization-promoting moieties include peroxidases such as horseradish peroxidase, and heme compounds such as hemin.

  The first reagent used in this step has a selective binding moiety to a molecule on the cell membrane and a radicalization promoting moiety. These moieties may be directly bonded or may be bonded by a linker group such as a peptide chain or an alkylene group. Examples of such a linker group include those similar to the linker group of Compound (I) described later. Examples of those in which these moieties are directly bonded include antibodies that are molecules on the target cell membrane and modified with radicalization promoting moieties. As the compound, a commercially available compound can be used, or it can be prepared by a conventional method using an HRP (horseradish peroxidase) labeling kit or the like.

  In order to cause the first reagent to act on cells, an aqueous solution of the compound is added to the cells and then incubated. The concentration of the aqueous solution of the first reagent may be adjusted as appropriate, but the concentration with respect to the culture solution is usually about 1 to 100 μg / mL. Incubation conditions may be appropriately adjusted in consideration of the type of cells to be used and the optimum temperature of the enzyme. Usually, the incubation conditions may be about 0 ° C. to 37 ° C. and about 10 minutes to 5 hours.

  After incubation, the cells are washed to remove excess first reagent. Washing may be repeated several times by removing PBS and adding PBS or the like and stirring gently.

(2) Second Step Next, the following compound (I) (hereinafter sometimes referred to as “second reagent”) is further allowed to act on the cells that have undergone the first step.
XYZN ₃ (I)
[Wherein, X represents a fluorescent fluorescein derivative group, Y represents a linker group, and Z represents an optionally substituted C _6-12 arylene group]

  The fluorescent fluorescein derivative group is not particularly limited as long as it is a fluorescein derivative and exhibits fluorescence. For example, the following can be illustrated.

The “linker group” is for facilitating the production of the compound (I) according to the present invention, that is, for binding the fluorescent fluorescein derivative group X and the azidoaryl group (—ZN ₃ ). In addition, the relatively bulky fluorescent fluorescein derivative group X also serves to prevent the binding of radicalized azido groups to the compound.

The “linker group” is not particularly limited as long as it has the above-described action. For example, a C _1-6 alkylene group, an amino group (—NR— (R represents a hydrogen atom or a C _1-6 alkyl group). )), Ether group (—O—), thioether group (—S—), carbonyl group (—C (═O) —), thionyl group (—C (═S) —); and C _1-6 alkylene And a group in which two or more groups selected from the group consisting of a group, an amino group, an ether group, a thioether group, a carbonyl group, and a thionyl group are linearly bonded.

Compound (I) (second reagent) can be easily produced by combining a fluorescent fluorescein derivative group X and an azidoaryl group (—ZN ₃ ) using methods known to those skilled in the art. For example, fluorescein and substituted with isothiocyanate and N- hydroxysuccinimide, are commercially available, such as Alexa (R) 488 is a fluorescein derivative substituted in likewise N- hydroxysuccinimide, these substituents -NH ₂ Since the compound can be easily bonded to a group, compound (I) can be produced by reacting the compound with an aryl azide substituted with a substituent having a —NH ₂ group at the tip.

  In this step, compound (I) (second reagent) is further allowed to act on the cells on which the first reagent has been allowed to act. In this step, the azide group of the second reagent is radicalized by the radicalization-promoting moiety of the first reagent selectively bound to the target compound, and binds to a nearby compound.

  Compound (I) (second reagent) may be added to the cells in a solution state. Specifically, an aqueous solution of about 10 to 100 μg / mL may be used. When compound (I) is difficult to dissolve in water, an organic solvent such as dimethyl sulfoxide or ethanol may be added to the extent that it does not adversely affect the cells used.

  The second reagent radicalized by the radicalization promoting moiety of the first reagent bound to the molecule on the target cell membrane is close to the molecule on the target cell membrane because of its high reactivity and the short distance that it can move as a radical. Bind to molecules only. For example, it is known that the migration distance of hydroxy radicals generated by the laser molecule inactivation method is a maximum of about 1.5 nm in radius, and the migration distance of singlet oxygen radicals generated by the fluorophore-assisted light inactivation method is about 10 to 50 nm. (J. C. Liao et al., Proc. Natl. Acad. Sci. USA, 91, 2659 (1994); S. Beck et al., Proteomics, 2, 247 (2002), etc.). Moreover, when the present inventors conducted another experiment using colloidal gold particles and an electron microscope, it was suggested that radicals generated by horseradish peroxidase exist within 300 nm from the probe molecule. From the above knowledge, it is considered that radicals generated by the reaction according to the present invention can bind to molecules existing within 300 nm from the target cell membrane molecule, but do not bind to molecules present at a position farther than that.

  The reaction conditions may be appropriately adjusted. For example, the reaction can be performed at 0 ° C. to 37 ° C. for about 5 minutes to 1 hour. Moreover, in order to suppress radicalization by light, the reaction is preferably performed in a dark place.

  After the reaction, excess reagent is removed by washing. More specifically, the operation of gently stirring with PBS or the like after removing the supernatant may be repeated several times.

(3) Third Step In this step, the compound to which the radicalized compound (I) (second reagent) is bound is specified. More specifically, first, the cells are physically disrupted with a homogenizer or the like. As a result, the cell membrane is finely cut into vesicles having a diameter of about 100 nm called microsomes. The microsome is separated from the nucleus by centrifugation or the like, and the obtained microsome fraction is lysed by lysis buffer or the like.

  Next, using the obtained lysate, a compound labeled with the second reagent is identified by a conventional method such as Western blotting, antibody array, mass spectrometry, immunoprecipitation, immunohistochemical staining, or the like.

  As a more specific method, for example, first, a compound contained in the obtained solution is separated. A specific separation method may be appropriately selected from methods generally used in the biochemical field. For example, a compound capable of separating compounds by molecular weight such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or gel filtration chromatography, a microarray to which an antibody is bound, or the like can be used.

  After separating the compounds, the compound to which compound (I) (second reagent) is bound is identified. For example, analysis may be performed at a fluorescence wavelength specific to the fluorescent fluorescein derivative group used, or analysis may be performed using a labeled antibody specific for the fluorescent fluorescein derivative group.

The kit of the present invention is a kit for carrying out the above-described method of the present invention,
A compound having a selective binding moiety to a molecule on a target cell membrane and a radicalization-promoting moiety, and a compound represented by the formula (I) XYZN ₃ (I)
[Wherein, X represents a fluorescent fluorescein derivative group, Y represents a linker group, and Z represents an optionally substituted C _6-12 arylene group]
It is characterized by including.

  In the kit of the present invention, the same compounds and specific means as those described in the method of the present invention can be used.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

  Example 1 Preparation of fluorescein labeled aryl azide

  4-Azidosalicyl (ω-aminohexamethylene) amide (33 mg, ASAHA) was dissolved in DMF (5 mL). A solution of fluorescein isothiocyanate (40 mg, Sigma, FITC) dissolved in DMF (5 mL) was added to the solution. The reaction mixture was allowed to react at room temperature for 12 hours while shielding light. After completion of the reaction, unreacted ASAHA was removed using a cation exchange resin (1 mL, manufactured by Bio-Rad, AG-50WX8). Fractions containing fluorescein-labeled aryl azide were collected and confirmed to have ASAHA removed by thin layer chromatography. The fraction was stored at −20 ° C. until use. Hereinafter, the obtained fluorescein-labeled aryl azide may be abbreviated as “FLA” (Fluorescein-Labbeled Arylazide).

  Comparative Example 1 Production of NBD-labeled aryl azide

  ASAHA (14 mg) was dissolved in DMF (5 mL). To the solution, a solution of 4-fluoro-7-nitrobenzofurazan (NBD, 3.1 mg, manufactured by Sigma) dissolved in DMF (5 mL) was added. Thereafter, a solution of NBD-labeled aryl azide was obtained in the same manner as in Example 1. Hereinafter, the obtained NBD-labeled aryl azide may be abbreviated as “NLA” (NBD-Labbeled Arylazide).

Example 2 Implementation of the method of the present invention (EMARS (Enzyme-Mediated Activation of Radical Source) reaction) Human cervical cancer cells (HeLa S3 cells) and human glioma cells (T98 human glioma cells) containing 5% CO ₂ The cells were cultured at 37 ° C. in an RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere.

  Separately, mouse hybridoma TS2 / 16 cells were cultured in ASF104 medium (Ajinomoto Co., Inc.). Anti-β1 integrin monoclonal antibody TS2 / 16 was purified from the supernatant of the medium using protein G-Sepharose (manufactured by Amersham Bioscience).

  The cells were washed once with PBS, 5 μg / mL PBS solution of the anti-β1 integrin monoclonal antibody TS2 / 16 was added, and the cells were treated at room temperature for 20 minutes. Further, a 5 μg / mL PBS solution of an anti-mouse IgG secondary antibody (Promega) conjugated with horseradish peroxidase (HRP) was added and treated at room temperature for 20 minutes. Further, as a comparative sample (negative control sample), a sample was prepared that was treated in the same manner except that the anti-β1 integrin monoclonal antibody TS2 / 16 was not used. After washing the cells, the 0.1 mM PBS solution of FLA produced in Example 1 above was added and incubated at room temperature for 15 minutes in the dark. The cells were then washed twice with PBS and collected in a plastic tube containing 100 mM Tris-HCl (500 μL). The cell suspension was disrupted by homogenizing the cell suspension with a syringe needle. Cell nuclei were precipitated by centrifuging the obtained cell disruption solution at 800 g for 5 minutes. Further, the supernatant was centrifuged at 20,000 g for 15 minutes to precipitate a microsomal fraction containing cell membrane components. Each precipitate was washed with 100 mM Tris-HCl (pH 7.4) and then dissolved with a lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 5 mM EDTA, 1% NP-40, 10% glycerol). did.

  Subsequently, after subjecting each sample to SDS-PAGE (gel concentration: 8% or 10%, non-reducing conditions), the gel was blotted onto a PVDF membrane, and goat anti-FITC antibody (manufactured by Rockland) and HRP conjugate. After treatment with an anti-goat IgG antibody (Santa Cruz), it was detected with immobilon Western blotting chemiluminescence detection reagent (Millipore). The results are shown in FIG.

  As shown in FIG. 1, in the microsomal fraction (fraction obtained by centrifugation at 20,000 g), when no anti-β1 integrin monoclonal antibody was used (negative control sample), almost no band was observed. On the other hand, in an EMARS (Enzyme-Mediated Activation of Radical Source) reaction sample using an anti-β1 integrin monoclonal antibody, a protein band that is considered to interact with β1 integrin, which is a membrane protein, has been detected. On the other hand, in the cell nuclear fraction (fraction obtained by centrifugation at 800 g), only a partial band is observed in human glioma cells, and non-specific binding of the second reagent according to the present invention is almost observed. Proven not.

Example 3 Dynamics of Second Reagent According to the Present Invention Human cervical cancer cells (HeLa S3 cells) were cultured for 16 hours on a 35 mm glass bottom plate (manufactured by Matsunami Glass Kogyo Co., Ltd.). The cultured cells were added with a 0.1 mM PBS solution of FLA produced in Example 1 above, and then treated at 4 ° C., room temperature or 37 ° C. for 15 minutes.

  Separately, human B-cell lymphoma cells (Daudi human B-cell lymphoma cells) were collected in a plastic tube, added with a 0.1 mM PBS solution of FLA prepared in Example 1 above, and then treated in the same manner as described above.

  The treated cells were carefully washed with PBS and observed with a confocal laser scanning microscope (OLYMPUS, FLUOVIEW FV1000). The results with human cervical cancer cells are shown in FIG. 2A, and the results with human B cell lymphoma cells are shown in FIG. 2B.

  As shown in FIG. 2, it is clear that in both human cervical cancer cells and human B-cell lymphoma cells, the second reagent according to the present invention (FLA in Example 1) is incorporated more into the cells as the temperature increases. It was. In Example 2 above, non-selective binding is suppressed according to the method of the present invention, and the reason why a compound that interacts with the target cell membrane molecule can be detected with high accuracy is that the second reagent is difficult to be taken into the cell initially. I thought. However, it was an unexpected result that the second reagent according to the present invention did not cause non-selective binding without being influenced by the endogenous enzyme in the cell while being taken into the cell.

Example 4 Implementation of the method of the present invention (EMARS reaction) Human cervical cancer cells (HeLa S3 cells) or human glioma cells (T98 human glioma cells) treated in the same manner as in Example 2 above were subjected to SDS-PAGE (gel concentration: (8% or 10% under non-reducing conditions) and then directly analyzing the gel in fluorescence mode using LAS-4000 Bio-imaging analyzer (Fuji Film) equipped with blue light and Y515-Di filter did. After analysis with LAS-4000, the gel was stained with Coomassie Brilliant Blue (CBB) G-250 (Nacalai Tesque). The results with human cervical cancer cells are shown in FIG. 3, and the results with human glioma cells are shown in FIG.

  As shown in FIGS. 3-4, when stained with CBB (Coomassie Brilliant Blue) that stains all protein bands, anti-β1 integrin monoclonal antibody was used (EMARS reaction sample) and not used (negative control sample). Since no difference was observed, it was confirmed that the same amount of protein was separated by SDS-PAGE in both samples. On the other hand, in order to specifically observe the band of the EMARS reaction product according to the present invention, when the band is detected by a fluorescence imager corresponding to the second reagent according to the present invention (FLA of Example 1), A band not observed in the negative control sample was strongly detected. Thus, according to the method of the present invention, a compound that interacts with β1 integrin, which is a molecule on the cell membrane, can be specifically labeled with the second reagent (FLA) and can be analyzed with a simple detection device called a fluorescence imager. Has been demonstrated.

Example 5 Implementation of the method of the present invention (EMARS reaction) In Example 2 above, instead of the combination of anti-β1 integrin monoclonal antibody TS2 / 16 and HRP-conjugated anti-mouse IgG antibody, HRP-conjugated CTxB (LIST Biological Laboratories) Human cervical cancer cells (HeLa S3 cells) were treated in the same manner except that 2 μg / mL PBS solution was used. CTxB is the B subunit of cholera toxin and specifically recognizes the glycosphingolipid GM1 present in the cell membrane.

  Subsequently, the obtained sample was subjected to SDS-PAGE (gel concentration: 8% or 10%, in a non-reducing environment), and then LAS-4000 Bio-imaging analyzer (Fujitsu) equipped with blue light and Y515-Di filter. The gel was directly analyzed in fluorescence mode. After analysis with LAS-4000, the gel was stained with Coomassie Brilliant Blue (CBB) G-250 (Nacalai Tesque). The results are shown in FIG.

  As shown in FIG. 5, when stained with CBB (Coomassie Brilliant Blue) that stains all protein bands, the first reagent, HRP-conjugated CTxB, is used (EMARS reaction sample) and not used (negative control sample). Since no difference was observed between the two samples, it was confirmed that the same amount of protein was separated by SDS-PAGE in both samples. On the other hand, in order to specifically observe the band of the EMARS reaction product according to the present invention, when the band was detected with a fluorescence imager corresponding to the second reagent according to the present invention (FLA of Example 1), a negative control sample was obtained. In contrast, almost no bands are observed, whereas many bands are detected in the EMARS sample. Thus, according to the method of the present invention, a compound that interacts with the glycosphingolipid GM1, which is a molecule on the cell membrane, can be specifically labeled with the second reagent (FLA), and a simple detection device called a fluorescence imager can be used. It was demonstrated that it can be analyzed.

Comparative Example 2 EMARS Reaction Using NLA of Comparative Example 1 In Example 5 above, instead of the FLA of Example 1 according to the present invention having a fluorescein derivative group, NLA having an NBD that is a fluorescent chromophore group was used. Conducted the experiment in the same manner. The results are shown in FIG.

  As shown in FIG. 6, when NLA is used, unlike the case of FLA according to the present invention, there are many non-specific bands, which should be specifically detected when HRP-conjugated CTxB is used. The band of the compound that interacts with the glycosphingolipid GM1 could not be identified. That is, even when HRP-conjugated CTxB was used, only the same non-specific band as in the case where HRP-conjugated CTxB was not used (negative control sample) could be detected. It is thought that this is because, besides the relatively weak fluorescence characteristics of NBD, NLA does not have the ability to suppress non-specific binding like FLA, and therefore the EMARS product cannot be specifically detected. It is done.

Comparative Example 3 EMARS Reaction Using Biotin Compound In Example 2 above, in addition to human cervical cancer cells (HeLa S3 cells) and human glioma cells (T98 human glioma cells), human B cell lymphoma cells (Daudi human B- cell lymphoma cell), and instead of the FLA produced in Example 1, EZ-Link biotin-LC-ASA in which biotin is bound to the 4-azidosalicylamino group via a linker group is used as the second reagent. The experiment was conducted in the same manner except that it was used. FIG. 7A shows the results of human cervical cancer cells, FIG. 7B shows the results of human glioma cells, and FIG. 7C shows the results of human B cell lymphoma cells.

  7A to 7C, when a compound having a biotin group as a labeling group is used as the second reagent, there is no difference between when the first reagent is used and when it is not used, and nonspecific binding is suppressed. It became clear that it was not possible.

Example 6 Purification of compound using the method of the present invention (EMARS reaction) (1) Preliminary experiment A 10 mM DMF solution (5 μL) of FLA prepared in Example 1 above was added to bovine serum albumin (Nacalai Tesque, BSA) at 370 μM. Added to PBS solution. The mixed solution was irradiated with ultraviolet rays on ice for 15 minutes to radicalize the aryl azide group and to bind FLA to BSA. The mixed solution was diluted stepwise with BSA, and then subjected to SDS-PAGE (gel concentration: 10%, in a non-reducing environment), and a fluorescence imager (manufactured by Fuji Film, LAS-4000 Bio-imaging analyzer). A band was detected. The gel was also stained with Coomassie Brilliant Blue (CBB) G-250. The results are shown in FIG.

  As shown in FIG. 8, the detection limit of the FLA-conjugated BSA fluorescence imager according to the present invention was 25 ng, which was almost the same as the detection limit by CBB staining. From these results, it was found that the EMARS reaction product according to the present invention exists at least on the order of nanograms as long as it can be detected by a fluorescence imager.

(2) Purification of EMARS reaction product Human cervical cancer cells (HeLa S3 cells) were cultured on a 15 cm plate, and without or using HRP-conjugated CTxB, as in Example 5 above. EMARS reaction was performed using FLA. After washing the microsomal fraction with 100 mM Tris-HCl (pH 7.4), a mixed solvent of chloroform: methanol = 2: 1 was added and gently stirred. Further, deionized water (500 μL) was added and gently stirred. This was centrifuged at 10,000 g for 5 minutes to separate the organic phase and the aqueous phase, and both the organic phase and the aqueous phase were removed leaving the microsomal fraction present in the intermediate layer. The remaining microsomal fraction was washed with 50% aqueous methanol (1 mL), and then centrifuged in the same manner to remove the liquid phase other than the precipitated microsomal fraction. After this operation was repeated three times, the precipitated microsomal fraction was dried under reduced pressure using an evaporator. To the obtained microsomal fraction, 100 mM Tris-HCl (pH 7.4) containing 1% of SDS was added and heated at 100 ° C. for 5 minutes. After removing insoluble matters, 400 μL of lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 5 mM EDTA, 1% NP-40, 10% glycerol) was added. To this lysate, a biotin-labeled goat anti-fluorescein antibody (Rockland, 20 μg) was added and incubated at 4 ° C. for 1 hour. Then, Dynabeads M-280 streptavidin (manufactured by Invitrogen) was added, and the compound having fluorescein was immunoprecipitated by incubating again at 4 ° C. for 1 hour. Dynabeads to which FLA according to the present invention was bound were washed five times with the above lysis buffer, twice with PBS containing 0.5 M NaCl, and once with distilled water. Next, the adsorbed FLA was eluted with non-reducing SDS-PAGE sample buffer at 50 ° C. for 15 minutes. The eluate was subjected to SDS-PAGE (gel concentration: 10%). In order to examine whether these series of immunoprecipitation experiments were successful, 5% volume of the lysate before the immunoprecipitation experiment and 5% volume of the non-adsorbed fraction after the immunoprecipitation experiment were collected. The sample was subjected to SDS-PAGE in the same manner as the immunoprecipitation sample (IP). After SDS-PAGE, analysis was performed with a fluorescence imager. Further, silver staining was performed using a Sylbest stain neo silver staining kit (manufactured by Nacalai Tesque). The results are shown in FIG.

  As shown in FIG. 9, as a result of analysis with a fluorescence imager, the sample (IP) obtained by immunoprecipitation of the lysate when HRP-conjugated CTxB was used (EMARS reaction product) did not use HRP-conjugated CTxB (negative) In comparison with the control sample, a plurality of specific bands relating to the compound that interacts with the glycosphingolipid GM1, which is a molecule on the cell membrane, were detected. Further, in the lysate before immunoprecipitation when HRP-conjugated CTxB was used, the same band as the immunoprecipitation sample (IP) was detected, but it was thin. Furthermore, those bands disappeared in the non-adsorbed fraction after immunoprecipitation. In addition, as a result of silver staining capable of detecting bands of all proteins, the number of silver staining bands in the immunoprecipitation sample (IP) was smaller than that in the lysate before the immunoprecipitation treatment, and these bands were detected by a fluorescence imager. It was found to be in the migration position corresponding to the band detected in (1).

  From the above results, by combining immunoprecipitation treatment with the method of the present invention, it is possible to purify FLA-conjugated molecules, that is, many compounds interacting with the glycosphingolipid GM1 present in the cell membrane, and simultaneously stain with silver staining. It was found that it can be concentrated to an appropriate amount. The amount of protein that can be silver-stained is about the same as the level at which protein can be identified by mass spectrometry.

Claims (4)

A method for detecting a compound that interacts with a molecule on a cell membrane,
Allowing a compound having a selective binding moiety to a molecule on a target cell membrane and a peroxidase or heme compound as a radicalization promoting moiety to act on a cell;
Further allowing the compound represented by formula (I) to act on cells;
XYZN ₃ (I)
[Wherein, X represents a fluorescent fluorescein derivative group, Y represents a linker group, and Z represents an optionally substituted C _6-12 arylene group]
A method comprising identifying a compound to which compound (I) is bound. The method of claim 1 fluorescent fluorescein derivative group is a fluoride les fluorescein group. The method according to claim 1 or 2 , wherein the compound to which compound (I) is bound is identified using SDS-PAGE. A kit for carrying out the method according to any one of claims 1 to 3
A compound having a selective binding moiety to a molecule on a target cell membrane, a peroxidase or a heme compound as a radicalization-promoting moiety, and a compound represented by formula (I) XYZN ₃ (I)
[Wherein, X represents a fluorescent fluorescein derivative group, Y represents a linker group, and Z represents an optionally substituted C _6-12 arylene group]
A kit comprising: