WO2007102396A1

WO2007102396A1 - Method for production of labeled sphingolipid

Info

Publication number: WO2007102396A1
Application number: PCT/JP2007/053917
Authority: WO
Inventors: Makoto Ito; Ai Yoshida; Hatsumi Monjusyo; Nozomu Okino
Original assignee: Kyushu University; Takara Bio Inc.
Priority date: 2006-03-02
Filing date: 2007-03-01
Publication date: 2007-09-13

Abstract

Disclosed is a method for production of a labeled sphingolipid, comprising reacting a sphingolipid or lysosphingolipid with an aliphatic carboxylic acid having a label selected from the group consisting of a substance capable of forming a chromophore, a fluorescent substance, biotin and a hapten by using sphingolipid ceramide N-deacylase to produce the labeled sphingolipid. The method enables to prepare any labeled sphingolipid including labeled sphingoglycolipid and labeled sphingomyelin with high efficiency and at a low cost.

Description

Specification

Method for producing labeled sphingolipid

Technical field

The present invention relates to a method for producing labeled sphingolipid useful for medicine, carbohydrate engineering, cell engineering, and the like, and a labeled sphingolipid obtained by the production method.

Background art

[0002] Sphingolipid is a general term for lipids having long-chain base sphingoids, including glycosphingolipids, sphingophospholipids, and ceramides, and is widely distributed from lower animals to higher animals. In recent years, these sphingolipids have been revealed to play an important role in biological activities such as cell proliferation, differentiation induction, and apoptosis. Moreover, since it is a structural component of the cell surface layer, it is also being used as an additive to cosmetics and the like.

[0003] The sphingolipid has a ceramide structure in which a long-chain fatty acid having a heterogeneous chain length is bound to an amide group of a sphingoid as an amide bond as a common structure. In general, sphingolipids do not exhibit characteristic absorption in the ultraviolet region or visible region, and are therefore difficult to detect optically by themselves. Therefore, labeling sphingolipids with chromophores, fluorescent dyes, piotin, antibody epitopes (no, pentene), etc., will help understand the functions of sphingolipids and biological membranes, leading to the development of drug discovery and artificial biological membranes. Is very useful for.

[0004] A long-chain fatty acid of a sphingolipid is modified! /, A method for producing a substituted sphingolipid or a sphingolipid derivative is based on a lysosphingolipid that lacks an acid amide-bonded fatty acid to the amino group of the sphingolipid of the sphingolipid. As a result, chemical and enzymatic synthesis methods are known.

[0005] As a chemical method, there is a method in which a fatty acid or a fatty acid derivative is condensed to an amino group of a lyso form by the following method. For example, a method using a fatty acid active ester such as N-hydroxysuccinimide ester of fatty acid, a method using a coupling reagent such as fatty acid and carbonyldiimidazole dicyclohexyl carpositimide, a method using a fatty acid anhydride, a fatty acid salt A method using a bowl is known. Non-patent Document 1, Patent Document 1 and Patent Documents use lysogandarioside as a lysate of acidic glycolipid. Reported in tribute 2. Further, it is described in Non-Patent Document 2 for the method power using sphingosyl phosphorylcholine (lysosphingomyelin) as a lyso form of sphingophospholipid. According to these methods, side reactions such as O-acylation may occur, and in order to obtain a product that is selectively N-acylated, complicated operations are required for the use and purification of protecting groups. It is. In addition, a sphingoid such as ceramide lysogandarioside obtained by chemically dehydrating a sphingoglycolipid containing a ceramide syratin and an amino sugar, a kind of sphingophosphonolipid. To selectively acylate only the sphingoid amino group of a sphingolipid having an amino group in addition to the amino group, introduction of a protecting group, partial acylation, partial deacylation after acylation, or an — After incorporating N-acetylylazogandarioside into the ribosome, complicated operations such as selective N-acetylation are necessary and difficult.

On the other hand, the enzymatic synthesis method is described in Patent Document 3. This method is a method in which condensation is carried out by lipase in an organic solvent, and a substantially anhydrous organic solvent is required, and the substrate is limited by the solubility of the substrate. In Patent Document 3, the force reaction that describes the enzymatic synthesis method of ceramide and hybrid ceramide is not specific, and the generation of O-acylates has been found. As in the case of having multiple amino groups, it is difficult to specifically act only on the amino group of the sphingoid. Non-Patent Document 3 and Patent Document 4 also describe the use of sphingolipid ceramide N-deacylase (SCD ase), an enzyme that specifically hydrolyzes acid amide bonds between sphingolipids of sphingolipids and fatty acids. Describes a method for producing a labeled sphingolipid by introducing a chromophore-forming substance, a fluorescent substance, piotin, a radioisotope, or the like into the fatty acid portion of the phospholipid. However, in the method disclosed herein, first, the ω-amino group of a ω-amino fatty acid protected by Ν-trifluoroacetylation is protected with lysosaccharide using SCDase derived from Pseudomonas bacteria. After introduction into the lipid, it is necessary to carry out complicated operations such as protection, deprotection and purification, such as deprotecting the Ν-trifluoroacetyl group and finally introducing a fluorescent dye into the ω-amino group. . In the above method, it has not been clarified whether a fatty acid to which a substance forming a chromophore or a fluorescent substance can be directly introduced into lysosphingolipid. [0007] Sphingolipid ceramide N-deacylase is known to be SCDase (Patent Document 5, Non-Patent Document 4) derived from the marine bacterium, Shewanella alga G8. Only C14 labeled fatty acids have been identified.

Patent Document 1: JP-A-2-200697

Patent Document 2: Japanese Patent Laid-Open No. 7-309888

Patent Document 3: International Publication No. 94Z26919 Pamphlet

Patent Document 4: Pamphlet of International Publication No.98Z03529

Patent Document 5: International Publication No. 2002Z026963 Pamphlet

Non-Patent Document 1: Methods in Enzymology, 138, 319-341 (1987)

Non-Patent Document 2: Journal of Lipid Research, 28th, 710-718 (1987)

Non-Patent Document 3: Journal of Biochemistry, Vol. 126, pp. 604-611 (1999)

Non-Patent Document 4: Journal of Biologi cal Chemistry, 277, 17300-17307 (2002)

Disclosure of the invention

Problems to be solved by the invention

[0008] As described above, the conventional method of chemically or enzymatically modifying or substituting long-chain fatty acids of sphingolipids to synthesize labeled sphingolipids is desirable because it produces by-products or limits the substrate. Or requires complicated operations. Therefore, an object of the present invention is to provide a production method for specifically and simply synthesizing a labeled sphingolipid obtained by modifying or substituting a lysosphingolipid or a long-chain fatty acid that binds to the sphingoid from the sphingolipid. Another object of the present invention is to provide a labeled sphingolipid obtained by the production method.

Means for solving the problem

[0009] The present inventors have conducted a study on a method for synthesizing a sphingolipid having a label, Binding of fatty acid with label to lysosphingolipid sphingoid amino group, or substitution power of fatty acid with acid amide bond to sphingoid of sphingolipid and fatty acid with other label Sphingoid acid amide bond of sphingoid The present inventors have found that sphingolipids having an arbitrary label can be synthesized by being catalyzed by SCDase, an enzyme that acts on lysosphingolipids and hydrolyzes into fatty acids. Conventionally, when preparing labeled sphingolipids by reverse reaction or transfer reaction with SCDase, ω-amino fatty acid having a protecting group is introduced into sphingolipids or lysosphingolipids using the above enzyme, followed by deprotection and labeling. Helped to react. However, the present inventors can directly introduce a fatty acid having a label on the sphingoid amino group of lysosphingolipid under mild conditions in an aqueous solution by using a specific SCDase, Alternatively, the present inventors have found that a labeled sphingolipid can be synthesized by a simple operation by directly exchanging a fatty acid having an acid amide bond with a sphingolipid of a sphingolipid with a fatty acid having another label.

That is, the present invention

[1] Using a sufingolipid ceramide N-deacylase, a sphingolipid or lysosphingolipid and a chromophore-forming substance, a fluorescent substance, an aliphatic carboxylic acid having a label selected from the group consisting of piotin and stencil A labeled sphingolipid, characterized in that the labeled sphingolipid is obtained,

[2] The production method according to the above [1], which is derived from a bacterium of the genus Sufingolipidceramide N-deacylase Cashebanella (Shewanella),

[3] The process according to the above [2], which is derived from Sufingolipidceramide N-deacylase Cassiabanara alga G8 strain,

[4] The production method of the above-mentioned [1], wherein Sphingolipidceramide N-deacylase is a recombinant enzyme,

[5] The production method according to the above [1], further comprising the step of isolating the obtained labeled sphingolipid, and

[6] The present invention relates to a labeled sphingolipid obtained by the production method according to any one of [1] to [5]. The invention's effect

[0011] The present invention provides a method for producing labeled sphingolipids efficiently in a simple process. INDUSTRIAL APPLICABILITY The present invention can directly introduce a fatty acid having a label into sphingolipid or lysosphingolipid, and is useful for industrial production of labeled sphingolipid.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be specifically described. In the present specification, “labeled sphingolipid” refers to a labeled sphingolipid.

In the present specification, the sphingolipid is a simple substance or a mixture of natural or synthetic products having a long-chain base sphingoid, including glycosphingolipid, sphingophospholipid, and ceramide. Can be mentioned. In the present specification, lysosphingolipid or lysozyme of sphingolipid refers to an N-deacyl form of sphingolipid lacking a fatty acid bonded with an acid amide to the amino group of sphingoid.

The sphingolipid that can be used in the present invention is not particularly limited to the present invention. For example, celebrity mouthside (GlcCer, GalCer, etc.), Gandario series (GM1, GM2, GDI, etc.), lacto series (ratatotetra) Osylceramide etc.), Neolacto series (Neolatatotetraosylceramide etc.), Sphingoglycolipids such as Globo series, and Sphingolin lipids such as Sphingomyelin, and these lyso forms can be used.

In the present specification, the aliphatic carboxylic acid includes a saturated fatty acid, an unsaturated fatty acid, and a hydrocarbon chain having a functional group such as a halogen, a substituted or unsubstituted amino group, an oxo group, a hydroxyl group, and a thiol group. Carboxylic acids having aliphatic properties such as substituted saturated or unsaturated fatty acids, or saturated or unsaturated fatty acids in which the hydrocarbon chain has elements such as oxygen, sulfur and nitrogen are included. Although it does not specifically limit the present invention, it is about C6-C26, preferably C10-C22, more preferably a saturated fatty acid having a carbon chain length of C12-C18, about C6-C26, preferably C10-C22, and more. Fatty acids such as unsaturated fatty acids having a carbon chain length of C12 to C18 and 2-hydroxy fatty acids are exemplified as the aliphatic rubonic acid used in the present invention. By using those fatty acids labeled with these fatty acids, labeled sphingolipids can be produced. [0016] The label used in the present invention means a substance capable of facilitating detection of a substance to which the label is added, and does not particularly limit the present invention, but forms a chromophore. It means substances, fluorescent substances, piotin, haptens, etc. As the substance that forms a chromophore, any commercially available chromogenic reagent can be used. Examples of such a chromophore include 4-phenol-trophenol, 2-phenol 1-phenol and 2-phenol. And naphthalene derivatives such as amino naphthalene. A commercially available fluorescent substance can be used as the fluorescent substance. Examples include 7—nitrobenz— 2—oxa— 1, 3 — diazole (NBD), 4, 4— difluor— 4— bora— 3a, 4a— diaza— s— indacene (B ODIPY (registered trademark)), fluorescein Not only these, but also a wide variety of fluorescent materials can be used. The hapten is not particularly limited as long as an antibody that recognizes the hapten is available. For example, digoxigenin can be used in the present invention. In the present invention, a fatty acid to which the above-mentioned label has been added (hereinafter sometimes referred to as “labeled fatty acid”), for example, NBD-labeled fatty acid, BODIPY-labeled fatty acid, or the like can be used. These labels can be easily introduced by reacting a derivative of a labeled compound having reactivity with an amino group or thiol group with a fatty acid having an amino group or thiol group. For example, examples of fluorescent substances and fluorescently labeled fatty acids that can be used in the present invention can be found in Invitrogen's Handbook of Fluorescent Probes and Research Products, Molecular Probes (Handbook of Fluorescent Prooes and Research Products, Molecular Probes). And it is self-loaded.

[0017] In the present invention, an enzyme that acts on an amide bond of a sphingoid of a sphingolipid and specifically hydrolyzes into a lysosphingolipid and a fatty acid, that is, a sphingolipid ceramide N deacylase (SCDase) is used. As a sphingolipid ceramid N deacylase suitable for use in the present invention, the ocean is an enzyme that acts widely on sphingolipids including sphingoglycolipids (gandariosides, neutral glycolipids) and sphingophospholipids (sphingomyelin). A force including SCDase (Non-Patent Document 4, Patent Document 5) derived from the strain Shewanella alga G8 is not limited thereto. Recombinant enzyme obtained using a gene encoding SCDase, and also encoding SCDase Even if it is a recombinant enzyme obtained by using a gene in which one or more, preferably 1 to 60, more preferably 1 to 30 bases are deleted, added, inserted or substituted in the base sequence of the gene, Any enzyme that specifically hydrolyzes the acid amide bond between lipid sphingoids and fatty acids is included in the SCDase referred to herein.

[0018] The gene encoding SCDase derived from Siebanella alga G8 strain is obtained from, for example, a DNA fragment inserted in a plasmid held by Escherichia coli JM109 ZpSE5 deposited under Patent Document 5 under the accession number FERM BP-7717. can do. Therefore, a recombinant SCDase can be produced using the above gene. Furthermore, recombinant SC Dase described in Patent Document 5 and Non-Patent Document 4 from which the C-terminal polypeptide is deleted can also be suitably used in the present invention.

[0019] In the method for producing a labeled sphingolipid of the present invention, in addition to the purified product of sphingolipid ceramide N-deacylase, a culture solution or a crude product containing the enzyme as long as a desired reaction can be catalyzed. An extract can also be used. Furthermore, when cultivating a microorganism having the ability to produce the enzyme, sphingolipid or lysosphingolipid and a labeled fatty acid may be added to the culture solution to produce labeled sphingolipid.

[0020] The labeled sphingolipid is produced by the method of the present invention in a buffer solution containing a raw sphingolipid or lysosphingolipid, a labeled aliphatic carboxylic acid, and SCDase. The amount of these raw materials used is not particularly limited and can be used up to the saturation amount. Usually, it is desirable that the aliphatic carboxylic acid is excessively present. However, in the present invention, the reaction proceeds even if the molar ratio of the sphingolipid or lysosphingolipid to the aliphatic carboxylic acid is 1: 1. Or lysosphingolipid may be present in excess. Therefore, it is desirable that the molar ratio of the sphingolipid or lysosphingolipid to the aliphatic carboxylic acid is 2: 1 to 1:10. Furthermore, the amount of the enzyme used is not particularly limited, and a wide range of force can be selected as appropriate. For example, per 1 mL of the starting solution, usually 0.1 lmU or more, preferably 0.5 mU to 200 mU, more preferably lmU to 10 mU, or The amount of sphingolipid or lysosphingolipid used is about 2 U to 20 U per lnmol.

[0021] As the buffer solution, a buffer solution having a pH of about 5 to 9 may be used in the present invention. It is desirable to carry out the production in a buffer solution of acid buffer or Tris-HCl buffer having a pH of around 6.5-9. Further, a surfactant may be usually added to the buffer solution for enzyme activity or substrate solubility. As the surfactant, the present invention is not particularly limited, but a bile acid surfactant, a nonionic surfactant, or the like can be used. The amount of surfactant to be added is particularly limited if it is set to an amount that activates the enzyme, dissolves the substrate, or is effective in obtaining the product efficiently. In the case of a nonionic surfactant, it is preferably added in the range of 0.01% to 2% by weight. More preferably, it is added in the range of 0.05% by weight to 0.5% by weight, and more preferably in the range of 0.05% by weight to 0.25% by weight. Metal salts may be added to the buffer to activate the enzyme! /. As the metal salt, for example, a divalent metal salt can be used, and for example, magnesium chloride, salt-manganese, salt-calcium and the like are preferably used. The addition amount of the metal salt is not particularly limited as long as the activity of the enzyme is recognized or the product can be obtained efficiently, but it is preferably in the range of 1 mM to 10 mM. It is preferable to add it with the following. Furthermore, an organic solvent may be added to these reaction solutions. The organic solvent at this time may be a water-soluble organic solvent, or the reaction may be carried out in a two-phase system formed using a water-insoluble organic solvent. The amount of organic solvent added is not particularly limited as long as the enzyme is not deactivated and the product can be obtained efficiently. The labeled sphingolipid thus produced can be confirmed, for example, by thin layer chromatography. The sphingolipid obtained according to the present invention is an unreacted sphingolipid or lysosphingolipid, and labeled aliphatic carboxylic acid by a purification means generally used for organic compounds, for example, extraction using an organic solvent or various chromatography. It can be separated and isolated and purified.

As described above, the present invention provides a method for producing labeled sphingolipid in which a label is added to the fatty acid of sphingolipid. In addition, the desired labeled sphingolipid can be industrially advantageously produced using the production method. Natural sphingolipids generally have a variety of long chain fatty acid chain lengths, and it has been difficult to obtain sphingolipids having a uniform long chain fatty acid chain length. However, by the method of the present invention, a sphingolipid having a chromophore-forming substance, a fluorescent substance, a marker such as piotin and the like, and a long-chain fatty acid homogenized Can be easily obtained. Since the presence and distribution of labeled sphingolipids obtained by the method of the present invention can be easily examined, detection of enzymes and proteins involved in sphingolipid metabolism, intracellular metabolism of sphingolipids, and elucidation of transport pathways It can be applied to

Example

[0023] Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited to the following examples.

[0024] Reference Example Preparation of Sphingolipid Ceramide N-Deacylase from Shewanella alga G8 Strain

Plasmid pSCDl was obtained from Escherichia coli JM109ZpSE5, which is a microorganism deposited under the accession number FERM BP-7717 described in Patent Document 5. PETSCD-del, a plasmid containing DNA encoding SCDase, which contains the DNA encoding SCDase contained in this plasmid and has a C-terminal polypeptide deleted in accordance with the procedure described in Patent Document 5. It was constructed.

[0025] Escherichia coli BL21 transformed with the plasmid pETSCD-del

(DE3) pLysE was cultured in LB medium, and cells were collected from the obtained culture solution. Sphingolipid ceramide N-deacylase (SCDase) was purified according to the following series of procedures described in Patent Document 5, using bacterial cells recovered from 15 liters of culture broth as a starting material.

(1) Fracture of cells by freeze-thawing and ultrasonic treatment

(2) Chromatography using HiTrap Chelating column (GE Healthcare Bioscience)

(3) Chromatography using HiTrap Q column (GE Healthcare Bioscience)

(4) Chromatography using HiLoad Superdex 200pg column (GE Healthcare Bioscience)

(5) Chromatography using HiTrap Q column (manufactured by GE Healthcare Bioscience) In addition, the activity of Sphingolipidceramide N-deacylase is as follows. It measured by the method of.

[0026] substrate solution [10 nmol gandarioside GMla, 5 mM MgCl, 5 mM Mn

2

CI, 5mM CaCl, 0. 2 weight ^_0/0 Triton X- 100, 200mM NaCl, 50mM acetate

twenty two

In the buffer solution (pH 5.5), 10 L of the crude enzyme solution was added and incubated at 37 ° C. for 30 minutes to react the crude enzyme solution with the substrate. Thereafter, the reaction was stopped by boiling at 100 ° C for 5 minutes. To the concentrated dried product prepared using the centrifugal concentrator, 10 L of black mouth form Zmethanol (2: 1, vZv) was added and sonicated to obtain the concentrated dried product. Dissolved. The resulting lysate was spotted on a silica gel TLC plate. Expanded with black mouth form Z methanol Z0.02% CaCl (5: 4: 1, vZvZv) and silica gel by orcinol sulfate method.

2

Glycolipids on the TLC plate were developed. Absorbance (wavelength: 540 nm) was measured using a TLC chromatographic scanner (manufactured by Shimadzu Corporation) on the spot on the silica gel TLC plate after color development, and the decomposition rate was measured by the following formula.

Decomposition rate (%) = ([area of free lysate GMla] Z [area of free lyso GMla + area of unreacted GMla]) X 100

Here, one unit (U) of SCDase was defined as the amount of enzyme that decomposes 1 μmol of GMla per minute. The optimum pH of the obtained SCDase was 5.5 to 6.5, and the molecular weight was 75 kDa.

Example 1 Preparation of lyso GMla

Gandarioside GMla (Sigma) 325 nmol (approx. 500 μg) and SC Dase 8.4 mU prepared in Reference Example 2.5 mM MgCl, 2.5 mM MnCl, 2.5 mM CaCl and 0.1

2 2 2 Weight ^_0/0 Triton (Triton) 25 mM sodium acetate buffer containing X- 100 at (pH 5. 5) in (in reaction volume 1200 37 After reacting 1 hour, the reaction solution 100 ° C The reaction was stopped by holding for 5 minutes, and the lyso-GMla was separated from this sample using an Oasis MCX column (Waters Co.) The Oasis MCX column was conditioned with methanol according to the manual attached to the product in advance and then with water. After equilibration, the reaction solution after the heat treatment was added to the column with hydrochloric acid so that the final concentration was 0.1 N. After washing the column with lm L of 0.1 N hydrochloric acid, 5 mL The adsorbate was eluted with 5 mL of methanol and 5 mL of 5% aqueous ammonia in methanol. (Thin layer chromatography) When analyzed with a plate (developing solvent: Kuroguchi form Z methanol ZO. 2% Ca CI solution = 2: 3: 1 (v / v / v), color reagent: orcinol sulfate) G

2

Mia was confirmed in the ammonia elution fraction. The solvent of this elution fraction was concentrated and removed to obtain purified lyso GMla.

Example 2 Condensation reaction of lyso GMla and C12—NBD—fatty acid

The lyso GM 1a obtained in Example 1 and 200 nmol of C 12- NBD fatty acid [7—nitroben z— 2—oxa— 1, 5-diazole (NBD) -labeled Laurie acid (also known as n—aodecanoic acid), Invitrogen The product was reacted at 37 ° C. for 1 hour in 25 mM Tris-HCl buffer pH 7.5 containing 0.5 wt% Triton X-100 supplemented with SCDase 5.6 mU prepared in Reference Example. The final volume of the reaction solution was 800. After completion of the reaction, the reaction product was purified by the method described in Example 1 using an Oasis MCX column. For each eluted fraction, TLC analysis was performed by the method described in Example 1. The results of TLC analysis are shown in Figs. Figure 1 shows the results of detecting reaction products by fluorescence under a UV lamp. Figure 2 shows the results of glycolipid detection for TLC plates using an orcinol sulfate reagent according to a conventional method. Lanes 1 and 2 in Figure 1 and 2 are samples applied to the column, lane 2 is a flow-through fraction, lane 3 is a hydrochloric acid wash fraction, lane 4 is a methanol elution fraction, lane 5 is aqueous ammonia Z methanol elution fraction, lane 6 Is an SCDase digest (marker) of Gandarioside GMla. It was confirmed that NBD-labeled GMla (C12—NBD-GMla) was recovered in the methanol elution fraction (lane 4).

The methanol elution fraction was further purified using Sep-Pak C18 and Sep-Pak plus Silica (Waters) and subjected to mass spectrometry. Mass spectrometry was performed in negative ion mode using API-3000 (Applied Biosystems). The sample was dissolved in 10 mM ammonium acetate solution Z-acetonitrile = 1: 1 solution (v / v) and introduced directly into the mass spectrometer. The resulting mask mouth matogram corresponds to a signal of mZz = 1640.3 corresponding to [M—H] —ion of C12—NBD—GMla (expected molecular weight 1 641) and [M—2H] ² —ion A signal of mZz = 819. 4 was confirmed.

From this result, it was shown that NBD-labeled fatty acid can be directly introduced into lyso GMla by using the method of the present invention. Example 3 Preparation of various fluorescent glycosphingolipids with SCDase

As glycolipid substrate, 1) Gandarioside GMla 2) Gandarioside Mitsutya 1) Galactosylceramide (GalCer) 4) Psychocin (Psy, galactosylsphingosine) was used. Gandarioside mistatia was prepared according to the method described in Methods in Enzymology, pages 14 and 660-664. GalCer (derived from bovine brain) and Psy were manufactured by Sigma-Aldrich. As fluorescently labeled fatty acids, A) C12—NBD—fatty acid B) C12—BODIPY—fatty acid used in Example 2 [(4, 4 difluor 4 bora 3a, 4a— diaza s—indacene (BODIPY) recognition Laurie acid, Manufactured by Invitrogen Co., Ltd. In each case, a final concentration of 250 M glycolipid substrate and fluorescently labeled fatty acid were prepared in the reference example, and SCDase was added to a final concentration of 0.5 mM Triton X-100 containing 25 mM Tris-HCl. The reaction was allowed to proceed for 16 hours at 37 ° C in buffer pH 7.5.The final volume of the reaction liquid was 20 μ. The amount of SCDase-added calorie i) 200 μ ii) 20 μ iii) 2.5 μ U. After completion of the reaction, the reaction solution was treated at 100 ° C for 5 min, concentrated to dryness, and then dissolved in 10 L of chloroform / methanol = 2: 1 (v / v), 5 μL of which was dissolved in TLC. Apply to the plate, mixed solvent black form Ζ methanol / 0.02% CaCl solution = 5: 4: 1 (V

2

ZvZv). The developed plate was observed under a UV lamp. When Gandarioside GM 1a was used, NBD-labeled GM 1a and BODIPY-labeled GM 1a were produced. When Gandarioside Mitastia was used. Is the ability to produce each NBD-labeled Gandarioside and BODIPY-labeled Gandarioside NB D-labeled GalCer and BODIPY-labeled GalCer-generated power When Psy is used NBD-labeled GalCer and BODIPY-labeled GalCer Generation was confirmed. The amount of fluorescent-labeled sphingoglycolipid produced increased as the amount of enzyme used increased. From these results, it was revealed that not only NBD-labeled fatty acids but also BO DIPY-labeled fatty acids were directly introduced into various glycosphingolipids by the method of the present invention. In addition, in the method of the present invention, in addition to the condensation reaction of the fluorescent labeled fatty acid acyl group directly with the amino group of the sphingosine base of the lysosulfoglycolipid, the sphingolipid is also exchanged by a reaction in which the fatty acid portion of the sphingolipid and the fluorescent labeled fatty acid are exchanged. A fluorescent group could be introduced into the glycolipid. That is, using either lysosphingoglycolipid or glycosphingolipid as a starting material (acceptor) It was also possible to introduce fluorescently labeled fatty acids (donors) into glycosphingolipids.

Example 4 Examination of reaction pH

In this example, lysogandarioside GMla was used as the acceptor glycolipid, and the C12-NBD-fatty acid used in Example 2 was used as the fluorescently labeled fatty acid. 25 mM buffer containing 0.1 wt% Triton X-100 and final concentration of 5 mM MgCl

2

The mixture was reacted at 37 ° C for 16 hours. The final volume of the reaction solution was 20. The buffer used was ρΗ4-6: sodium acetate buffer; pH 6-7.5: phosphate buffer; pH 7.5-9.5: Tris-HCl buffer. After completion of the reaction, the reaction solution was treated at 100 ° C for 5 minutes. The reaction solution was concentrated to dryness and then dissolved in 200 L of Kuroguchi Form Z methanol = 2: 1 (v / v), 5 L of which was applied to TLC. Chloroform formaldehyde / methanol / 0.02% CaCl = 5: 4: l (vZvZv)

2

The fluorescent lipid was quantified with OOPC (manufactured by Shimadzu Corporation). The results are shown in Figure 3. The condensation reaction rate was good in the range of pH 7.0 force to pH 9.0.

[0031] Example 5 Examination of surfactant

In this example, lysogandarioside GMla was used as the acceptor glycolipid, and the C12-NBD-fatty acid used in Example 2 was used as the fluorescently labeled fatty acid. 25 mM Tris HCl buffer containing SCDase 80 μ U prepared in the reference example and final concentrations of 5 mM MgCl, each containing a final concentration of 2,500 μΜ glycolipid and fluorescently labeled fatty acid, p

2

The mixture was reacted at 37 ° C for 16 hours in H7.5. The final volume of the reaction solution was 20. The surfactants used were Triton X-100 (nonionic), sodium cholate (anionic), sodium taurodeoxycholate (TDC, anionic). For each surfactant, the final concentration was changed to 0%, 0.05% by weight, 0.1% by weight, 0.25% by weight, 0.5% by weight, 1% by weight, and 2% by weight. After completion of the reaction, the product was analyzed by the method described in Example 4. As a result, in the reaction to which Triton X-100 was added, a good condensation reaction rate was obtained when the concentration was in the range of 0.05 wt% to 0.25 wt%.

Example 6 Examination of metal salt

In this example, lysogandarioside GMla is used as an acceptor glycolipid, and a fluorescent standard is used. As the fatty acid, the C12-NBD-fatty acid used in Example 2 was used. The final concentration of 0.1% by weight of Triton X-100 and the final concentration of 5 mM of various metal salts were prepared by adding SCDase 80 μU prepared in a reference example with a glycolipid with a final concentration of 250 μΜ each and a fluorescently labeled fatty acid. The reaction was carried out at 37 ° C for 16 hours in 25 mM Tris-HCl buffer, pH 7.5. The final volume of the reaction solution was 20 μm. The metal salts used are CaCl, MnCl, MgCl, CuCl, ZnCl.

2 2 2 2 2 We also examined EDTA. After completion of the reaction, the product was analyzed by the method described in Example 4. The results are shown in Fig. 4. Condensation rate is calculated by adding CaCl, MnCl, MgCl

2 2 2 was better than that without metal salt.

Example 7 Examination of enzyme amount

In this example, lysogandarioside GMla was used as the acceptor glycolipid, and the C12-NBD-fatty acid used in Example 2 was used as the fluorescently labeled fatty acid. Include each MgCl final concentration 2 50 M final concentration 0.1 wt ^_0/0 was added Caro the SCDase prepared in Reference Example and a glycolipid and a fluorescent-labeled fatty acids (5 nmol) Triton X-100 and a final concentration of 5mM 25 mM Tris

2

The reaction was carried out in hydrochloric acid buffer, pH 7.5, at 37 ° C for 16 hours. The final volume of the reaction solution was 20. The amount of enzyme used was 2000 U, 80 U, 40 U, 20 U, 10 μU, 5 U, 2.5 U, and 1.25 U, respectively. These are 400 U, 16 U, 8 U, 4 U, 2 U, 1 U, 0.5 U, and 0.25 U per lnmol of glycolipid, respectively. After completion of the reaction, the product was analyzed by the method described in Example 4. The results are shown in FIG. The condensation reaction rate was good when 20 μU to 80 μU SCDase was used.

Example 8 Labeling of sphingolipid

The following 1-11 sphingolipids were used as acceptors, and the reaction was carried out using the C 12 -NBD-fatty acid used in Example 2 as the fluorescently labeled fatty acid. A final concentration of 250 M glycolipids and fluorescently labeled fatty acids were prepared in the reference examples, and SCDase 40 U was added to a final concentration of 0.1 mM Triton X-100 and a final concentration of 5 mM CaCl containing 25 mM Tris HCl.

2

The reaction was carried out at 37 ° C. for 16 hours in a buffer, PH 7.5. The final volume of the reaction solution was 20 L. Sphingolipids used were 1) Sphingomyelin (derived from chicken egg yolk, manufactured by Sigma Aldrich), 2) Darcosylceramide (GlcCer, manufactured by Sigma-Aldrich), 3) Ratatosyl ceramide (LacCer, manufactured by Sigma-Aldrich) 4) Gandarioside GM3 (from Ushi brain) 5) Gandarioside GM2 (Matreya), 6) Gandarioside GMla (from Ushi brain), 7) Gandarioside GDla (from Ushi brain), 8) Gandarioside GTlb (from Ushi brain), 9) Ganglioside GQ lb 10) Crude Gandarioside Mixture (from porcine brain). After completion of the reaction, the product was subjected to TLC analysis by the method described in Example 4, and fluorescently labeled sphingolipid was detected under UV light. As a result, it was confirmed that all the sphingolipids used were fluorescently labeled.

Example 9 Labeling with Rhizogandarioside GMla Production of GMla

In this example, Rhizogandarioside GMla was used as the acceptor glycolipid, and C12-NBD-fatty acid used in Example 2 was used as the fluorescently labeled fatty acid. Final concentration 250 M (each 1 50 nmol) of the glycolipid substrate and a fluorescent-labeled fatty acid and a MgCl of SCDase 1. 2m U final concentration was added to 0.1 weight ^_0/0 Triton X- 100 and 5mM prepared in Reference Example 25mM including

2

The mixture was reacted at 37 ° C for 16 hours in a squirrel hydrochloric acid buffer, pH 7.5. The final volume of the reaction solution was 600 μm. After completion of the reaction, the reaction solution was treated at 100 ° C for 5 minutes, and labeled GMla was separated using an Oasis MCX column (manufactured by Waters). The Oasis MCX column was conditioned with methanol and equilibrated with water according to the manual supplied with the product. Hydrochloric acid was added to the reaction solution after heat treatment to a final concentration of 0.1 N, and then applied to the column. After washing the column with 2 mL of 0.01N hydrochloric acid, the adsorbate was eluted with 5 mL of methanol and 5 mL of 5% aqueous ammonia in methanol. Sample 1Z 50 from the elution fraction, remove the solvent, dissolve in 100 μL of black mouth form Ζ methanol = 2: 1 (ν / ν), 5 L of which is used for NBD detection, For glycolipid detection, each was applied to TLC plate and black mouth form ホル methanol Ζ0.02% CaCl

Two solutions were developed using 5: 4: 1 (vZvZv). The developed NBD detection plate was subjected to fluorescence detection using a chromatographic scanner CS9300PC according to a conventional method. The glycolipid detection plate was quantified by using orcinol sulfate as a coloring reagent. As a result, the condensation rate of the above reaction was about 40%, the purification recovery rate of the reaction product C12-NBD-GMla was about 90%, and the obtained C12-NBD-GMla was about 50 nmol.

Example 10 Preparation of fluorescently labeled sphingomyelin

In this example, Sphingomyelin (SM, Sigma-Aldrich) was used as the acceptor lipid. As a fluorescently labeled fatty acid, A) C12-NBD-fatty acid used in Example 2 or B) C12-BODIPY-fatty acid used in Example 3 was used. 25mM Tris, each containing a final concentration 250 M of the scan Fuingo SCDase 200 μ U or 2 0 mu final concentration 0.1 weight with the addition of U ^_0/0 Triton X- 100 for the lipids and fluorescent-labeled fatty acid prepared in Reference Example The reaction was carried out in a hydrochloric acid buffer, pH 7.5, at 37 ° C for 16 hours. The final volume of the reaction solution was 20. When TLC analysis of the reaction solution was performed by the method described in Example 3, it was confirmed that NBD-labeled SM and BOD IPY-labeled SM were generated. From this result, it became clear that the fluorescently labeled fatty acid can be introduced using sphingomyelin as an acceptor by the method of the present invention.

Industrial applicability

[0037] The production method of the present invention has made it possible to efficiently and inexpensively prepare any labeled sphingolipid containing sphingoglycolipid and sphingomyelin. The labeled sphingolipid obtained by the method of the present invention is useful for the development of medical technologies such as drug discovery and artificial biomembranes, as well as research in the fields of physiology and biochemistry such as elucidation of functions of sphingolipids and biomembranes. Brief Description of Drawings

FIG. 1 is a diagram showing the results (fluorescence signal) of TLC analysis of NBD-labeled GMla produced by the method of the present invention.

FIG. 2 is a diagram showing the results (sugar staining) of TLC analysis of NBD-labeled GMla produced by the method of the present invention.

FIG. 3 is a graph showing the results of examination of buffer solution and pH.

FIG. 4 is a diagram showing the results of study on metal salts.

FIG. 5 is a diagram showing the results of examination of the amount of enzyme.

Claims

The scope of the claims

[1] Sphingolipid ceramide N-deacylase is obtained by combining sphingolipid or lysosphingolipid with a chromophore-forming substance, a fluorescent substance, piotin, and an aliphatic carboxylic acid having a label selected from the group consisting of ptenca. A labeled sphingolipid production method, characterized in that the labeled sphingolipid is obtained by reacting with the use of

[2] The production method according to claim 1, wherein the bacterium is derived from a bacterium belonging to the genus Sheungella, Sphingolipidceramide N-deacylase.

[3] The production method according to claim 2, wherein the method is derived from Sufingolipidceramide N-deacylase Cassiva laarga G8.

[4] The production method according to claim 1, wherein the sulfingolipidceramide N-deacylase is a recombinant enzyme.

[5] The production method according to claim 1, further comprising the step of isolating the obtained labeled sphingolipid.

[6] A labeled sphingolipid obtained by the production method according to any one of claims 1 to 5.