JPWO2005097155A1 - Neurite outgrowth inducer - Google Patents

Abstract

Novel neurite outgrowth agent, therapeutic agent or preventive agent for neurodegenerative disease, food or beverage for treatment or prevention of the disease, method for inducing neurite outgrowth, treatment of neurological disease or Prophylactic methods, methods for improving or improving learning and / or memory ability, use of the sugar chain for the manufacture of a medicament for the treatment or prevention of neurological diseases, and production of agents for improving or improving learning and / or memory ability Providing the use of said sugar chain for

Description

  The present invention relates to a sugar chain having a neurite outgrowth action, a complex carbohydrate, the sugar chain or a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain or the complex carbohydrate, or the sugar chain The present invention relates to an enzyme for synthesis and a pharmaceutical composition, reagent, food or beverage comprising a nucleic acid encoding the enzyme as an active ingredient.

  A nerve cell usually constructs a complex network by extending a single axon and a plurality of dendrites and exchanging signals with other cells. A series of substances called neurotrophic factors play an important role in maintaining the functions of neurons, such as the survival of neurons, differentiation from juvenile neuroblasts into mature neurons, and neurite outgrowth. It is considered.

  Diseases involving nerve damage or neurodegeneration, such as nerve amputation, spinal cord injury, diabetic neuropathy, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Alzheimer's disease, Peripheral and central nervous system diseases such as Parkinson's disease and Huntington's disease can be differentiated into nerve cells and methods that promote the survival and functional repair of damaged neurons. It is broadly classified into a method (replenishment regeneration) for replenishing the function by supplementing cells (for example, neural stem cells) to the damaged site. Among these, the replenishment regeneration has not yet become widespread due to the difficulty in obtaining the replenished cells and the existence of ethical problems. On the other hand, the preservation regeneration is more specifically a method for regenerating the function by regenerating axons from the remaining nerve cells and forming new synapses.

  In recent years, neurotrophic factors have attracted attention for the purpose of preventing and treating neurodegenerative diseases. For example, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4 / 5, etc. have been found. Yes. These nerve growth factors are promising as therapeutic agents for neurological dysfunction caused by various neuronal cell disorders and preventive agents for dementia (for example, see Non-Patent Document 1).

  For example, Patent Document 1 reports a neurotrophic factor having a molecular weight of 60000 ± 5000 and a neurotrophic factor having a molecular weight of 120,000 ± 5000 obtained from an astrocytoma, glial cell, or Schwann cell culture medium. The neurotrophic factor described in Patent Document 1 exhibits a neuroblastoma growth inhibitory activity and also has a survival activity or neurite outgrowth action on nerve cells. Furthermore, Patent Document 2 reports a glycosaminoglycan derivative that has low anticoagulant activity and exhibits neurite outgrowth action.

  However, since these proteinaceous neurotrophic factors, polysaccharide derivatives and the like are high molecular weight compounds, it is extremely difficult to make the nutritional factors reach the affected area in the brain across barriers such as the blood-brain barrier. Therefore, it is a problem that the effects of the proteinaceous neurotrophic factor, polysaccharide derivatives and the like are greatly affected.

On the other hand, as a low molecular weight neurotrophic factor, for example, a neurite elongation agent containing a polyalkoxyflavonoid extracted from a citrus plant (Patent Document 3), a nerve cell growth action such as a nerve cell proliferation action and a neurite extension action A neurite elongation agent (Patent Document 5) containing a cyclohexenol derivative (Patent Document 4) and a pyrrolidine derivative or piperidine derivative exhibiting an promoting action has been reported. However, at present, these substances are not sufficiently effective.
JP 07-101990 A JP-A-11-310602 JP 2002-060340 A JP 2001-089404 A JP 2001-247469 A “Nature”, 344, 339-341 (1990)

  The present invention relates to a novel neurite outgrowth agent, a therapeutic agent or preventive agent for neurodegenerative diseases, a food or beverage for treatment or prevention of the disease, a method for inducing neurite outgrowth, a nerve, which exhibits a high neurite elongation effect Method for treating or preventing a disease, method for improving or improving learning and / or memory ability, use of the sugar chain for the manufacture of a medicament for treatment or prevention of a neurological disease and an agent for improving learning and / or memory ability or It relates to providing the use of the sugar chain for the production of an improving agent. In one aspect, the present invention relates to providing a neurite outgrowth inducer that can facilitate the formation of new neural networks based on neurite outgrowth. In another aspect, the present invention relates to providing a therapeutic or preventive agent for neurological diseases that can improve symptoms of diseases associated with neurodegeneration, suppress the onset or progression of the diseases, and the like. In yet another aspect, the present invention makes it possible to improve the learning and / or memory capacity of an animal individual, to perform the learning and / or restoration of the memory capacity of an individual that has been reduced due to various factors, etc. And / or providing a memory capacity improver or improver. In another aspect, the present invention relates to providing a food or beverage that can obtain a therapeutic or preventive effect for neurological diseases, an effect of improving or improving learning and / or memory ability, and the like by ingestion. In yet another aspect, the present invention relates to providing a method for inducing neurite outgrowth that allows inducing the generation and / or extension of neurites that differentiate into axons or dendrites. In another aspect, the present invention provides a method for treating or preventing a neurological disease, which can improve symptoms of a disease associated with neurodegeneration, suppress the onset or progression of the disease, and the like, and the neurological disease Provided the use of a defined sugar chain for the manufacture of a medicament for the treatment or prevention of a neurological disease, enabling the provision of a medicament suitable for performing the treatment or prevention method, the treatment or prevention of a neurological disease, etc. About that. Furthermore, in another aspect, the present invention enables the learning and / or memory ability of an animal individual to be increased, the learning and / or restoration of the memory ability of an individual that has been reduced due to various factors, etc. And / or a method for improving or improving memory capacity, and a learning and / or supply of a memory capacity improver or improver suitable for performing the learning and / or memory capacity improving or improving method, learning in an individual and / or The present invention relates to providing a use of a defined sugar chain for the production of an agent for improving and improving learning ability and / or improving memory ability, which enables improvement or improvement of memory ability and the like. In addition, the other subject of this invention is derived by description of this specification.

  That is, the first invention of the present invention is a sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, the sugar chain or a derivative of the complex carbohydrate, the sugar chain or the complex carbohydrate. The present invention relates to a neurite outgrowth inducer comprising as an active ingredient at least one selected from the group consisting of a pharmacologically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme.

  A second invention of the present invention is a sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a sugar chain or a derivative of the complex carbohydrate, a drug of the sugar chain or the complex carbohydrate The present invention relates to a therapeutic or prophylactic agent for neurological diseases containing as an active ingredient at least one selected from the group consisting of a physically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Examples of the neurological diseases include neurodegenerative diseases, dementia, brain tumors and the like.

  The third invention of the present invention is a sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a sugar chain or a derivative of the complex carbohydrate, a drug of the sugar chain or the complex carbohydrate An agent for improving learning and / or memory ability comprising, as an active ingredient, at least one selected from the group consisting of a physically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme, or It relates to an improving agent.

  The fourth invention of the present invention is a sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a sugar chain or a derivative of the complex carbohydrate, a drug of the sugar chain or the complex carbohydrate The present invention relates to a food or beverage containing at least one selected from the group consisting of a salt that is physically acceptable, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. In the fourth invention of the present invention, the food or beverage is a food or beverage used for the treatment or prevention of neurodegenerative diseases, dementia or brain tumors, the treatment or prevention of the neurodegenerative diseases, dementia or brain tumors. Examples include foods or beverages for assisting, foods or beverages used for improving or improving learning and / or memory ability, foods or beverages for assisting in improving or improving learning and / or memory ability, etc. .

  The fifth invention of the present invention is a sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in the structure, a derivative of the sugar chain or the complex carbohydrate, a drug of the sugar chain or the complex carbohydrate A method for inducing neurite outgrowth, comprising administering at least one selected from the group consisting of a physically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme to a specimen. .

  In the first to fifth inventions of the present invention, the sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The sugar chain represented by these is illustrated.

That is, the gist of the present invention is as follows.
[1] A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, A neurite outgrowth comprising, as an active ingredient, at least one selected from the group consisting of a pharmacologically acceptable salt of a complex carbohydrate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme Inducer,
[2] A sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
A neurite outgrowth inducer according to the above [1], which is a sugar chain represented by:
[3] A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
A neurite outgrowth inducer according to the above [1], which is a sugar chain represented by:
[4] A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, A neurological disease treatment comprising as an active ingredient at least one selected from the group consisting of a pharmacologically acceptable salt of glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme Agent or preventive agent,
[5] A sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The therapeutic agent or prophylactic agent for neurological diseases according to [4] above, which is a sugar chain represented by:
[6] A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
The therapeutic agent or prophylactic agent for neurological diseases according to [4] above, which is a sugar chain represented by:
[7] The therapeutic or preventive agent for neurological disease according to any one of [4] to [6], which is used for the treatment or prevention of neurodegenerative disease, dementia or brain tumor,
[8] A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, A pharmacologically acceptable salt of glycoconjugate, containing at least one selected from the group consisting of an enzyme involved in the synthesis of the sugar chain and a nucleic acid encoding the enzyme as an active ingredient, Or memory capacity improver or improver,
[9] A sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The learning and / or memory ability improving agent or improving agent according to the above [8], which is a sugar chain represented by:
[10] A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have GlcNAc residues, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
The learning and / or memory ability improving agent or improving agent according to the above [8], which is a sugar chain represented by:
[11] A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, and the A food or beverage comprising at least one selected from the group consisting of pharmacologically acceptable salts of complex carbohydrates,
[12] A sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The food or beverage according to the above [11], which is a sugar chain represented by
[13] A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
The food or beverage according to the above [11], which is a sugar chain represented by
[14] The food or beverage according to any one of [11] to [13], which is used for treatment or prevention of a neurodegenerative disease, dementia or brain tumor,
[15] The food or beverage according to any one of [11] to [13], which is used for improving or preventing learning and / or memory ability,
[16] A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, A nerve comprising administering to a specimen at least one selected from the group consisting of a pharmacologically acceptable salt of a complex carbohydrate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme A method of inducing protrusion extension,
[17] The sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The method for inducing neurite outgrowth according to the above [16], which is a sugar chain represented by
[18] A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
The method for inducing neurite outgrowth according to the above [16], which is a sugar chain represented by
[19] To an individual suffering from a neurological disease or an individual who is likely to suffer from the neurological disease, the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
A sugar chain having a bisecting GlcNAc represented by: a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, A neurological disease characterized by administering at least one selected from the group consisting of a pharmacologically acceptable salt of the glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Treatment or prevention methods,
[20] For individuals who need to improve or improve learning ability, the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. Indicates
A sugar chain having a bisecting GlcNAc represented by: a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, Supplying at least one selected from the group consisting of a pharmacologically acceptable salt of the glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme, and / Or a method for improving or improving memory capacity,
[21] The following formula (2) for the manufacture of a medicament for the treatment or prevention of neurological diseases:

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
A sugar chain having a bisecting GlcNAc represented by: a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, Pharmacologically acceptable salt of the glycoconjugate, use of at least one selected from the group consisting of an enzyme involved in the synthesis of the sugar chain and a nucleic acid encoding the enzyme, and [22] learning and / or Formula (2) for the production of a memory capacity improver or improver:

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have GlcNAc residues, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
A sugar chain having a bisecting GlcNAc represented by: a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, Use of at least one selected from the group consisting of a pharmacologically acceptable salt of the glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme,
About.

  The present invention provides a pharmaceutical, reagent, food or beverage containing a sugar chain having a bisecting GlcNAc, a complex carbohydrate, a derivative thereof and / or a salt thereof. That is, according to the neurite outgrowth inducer of the present invention, there is an excellent effect that the formation of a new neural network can be promoted based on the neurite outgrowth. Moreover, according to the therapeutic agent or preventive agent for neurological diseases of the present invention, it is possible to improve the symptoms of diseases associated with neurodegeneration, and to produce an excellent effect that the onset or progression of the diseases can be suppressed. . Furthermore, according to the learning and / or memory ability improving or improving agent of the present invention, the learning and / or memory ability of an animal individual can be increased, and the learning and / or memory of an individual that has decreased due to various factors. It has an excellent effect that memory ability can be recovered. According to the food or beverage of the present invention, there is an excellent effect that the effect of treating or preventing neurological diseases, the improvement of learning and / or memory ability, or the improvement effect can be obtained by ingesting the food or beverage. Furthermore, according to the method for inducing neurite outgrowth of the present invention, it is possible to induce the generation and / or extension of neurites that differentiate into axons or dendrites. According to the neurological disease treatment or prevention method of the present invention, it is possible to improve the symptoms of a disease associated with neurodegeneration and to suppress the onset or progression of the disease. Furthermore, according to the method for improving or improving the learning and / or memory ability of the present invention, the learning and / or memory ability of an animal individual can be increased, and the learning and / or memory of an individual that has decreased due to various factors. There is an excellent effect that the ability can be restored. In addition, according to the use of the defined sugar chain for the manufacture of a medicament for the treatment or prevention of a neurological disease of the present invention, the supply of a medicament suitable for carrying out the method for treating or preventing the neurological disease, Excellent effect of enabling treatment or prevention. Furthermore, according to the use of the defined sugar chain for the production of the learning and / or memory ability improving agent or improving agent of the present invention, it is suitable for performing the learning and / or memory capacity improving or improving method. There is an excellent effect that it is possible to supply an improving or improving agent for learning and / or memory ability, and to improve or improve learning and / or memory ability in an individual.

FIG. 1 is a diagram showing the neurite outgrowth-inducing action by Gn (Gn) Gn-bi-GP. In the figure, white bars indicate nerve extension, and black bars indicate cell extension.

FIG. 2 is a diagram showing neurite outgrowth in GnT-III gene-introduced cells.

  The present invention is based on the fact that a sugar chain having a bisecting GlcNAc (bisecting GlcNAc) structure, a complex carbohydrate having the sugar chain structure, and an enzyme related to the synthesis of the sugar chain exhibit an excellent neurite extension action. Based on surprising findings.

  In one aspect, the present invention relates to a neurite outgrowth inducer.

  The neurite outgrowth inducer of the present invention comprises a sugar chain having the bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, the sugar chain or a derivative of the complex carbohydrate, the sugar chain or the complex carbohydrate One major feature is that it contains, as an active ingredient, at least one selected from the group consisting of a pharmacologically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Therefore, according to the neurite outgrowth inducer of the present invention, a high neurite outgrowth inducing effect can be obtained at a site where neurite outgrowth is desired, such as a site of nerve damage, a site where neurodegeneration is observed, etc. It exhibits an excellent effect of efficiently extending neurites.

  In the differentiation of undifferentiated neurons, first, neurite elongation occurs, and then one of the neurites rapidly expands to show the properties of axons, and the rest Neurites acquire dendritic properties. The axons and dendrites of the cells generated in this way form synapses with other cells to construct a neural circuit. According to the neurite outgrowth inducer of the present invention, an axon or dendrite is generated from the induced neurite, and an excellent effect of promoting the formation of a new neural network is exhibited. Furthermore, according to the neurite outgrowth inducer of the present invention, it becomes possible to regenerate nerve cells, treat or prevent neurological diseases such as neurodegenerative diseases, etc. by extending the neurites.

In general, a sugar chain of a glycoprotein is composed of an N-glycoside-linked sugar chain (also referred to herein as “N-glycan”) bonded to an asparagine residue of the protein, a serine residue or a threonine residue of the protein. It is classified as an O-glycoside-linked sugar chain (also referred to as “O-glycan”) bonded through the hydroxyl group of the group. In the N-glycan, N-acetylglucosamine (GlcNAc), which is a reducing end sugar residue, is bonded to an amino group of asparagine on the peptide chain, and the anomeric hydroxyl group of the reducing end N-acetylglucosamine is removed, [ sugar _{-NH-CO-CH 2 -CH (} NH 2) COOH ] to form a coupling state of the form.

  N-glycans further include complex sugar chains composed of sugar residues such as sialic acid, galactose, mannose, fucose, N-acetylglucosamine (also referred to as “N-acetyllactosamine-type sugar chains”), mannose (Man) And N-acetylglucosamine oligomannose-type sugar chains, mixed-type sugar chains having a structure in which the N-acetyllactosamine-type sugar chains and oligomannose-type sugar chains are combined (also referred to as “hybrid-type sugar chains”) 3).

  The sugar chain having bisecting GlcNAc in the present invention is a sugar chain classified as the complex sugar chain or the hybrid sugar chain, and is bonded to the hydroxyl group at the 4-position of the β-mannose residue of the sugar chain. Examples thereof include sugar chains having N-acetylglucosamine.

  Examples of sugar chains having complex bisecting GlcNAc that can be used in the present invention include the following formula (1):

(In the formula, R represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
And the like.

  In addition, the sugar chain of the formula (1) is, in other words, the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ may be bonded to mannose (Man) via a β1-2 bond, a β1-4 bond, or a β1-6 bond. GlcNAc residue is shown (however, when R ² and R ³ have a GlcNAc residue, 1 to 3 R ² and R ³ may be directly bonded to mannose).
It is a sugar chain included in the sugar chain represented by.

  Specifically, for example, the following formula (3):

[Wherein, R ¹ represents an arbitrary sugar residue, R ² represents a GlcNAc residue which may be bonded to mannose (Man) via a β1-6 bond or a β1-4 bond, and R ³ represents a GlcNAc residue which may be bound to mannose (Man) via a β1-4 bond or a β1-6 bond.
The sugar chain represented by these is mentioned.

  As the sugar chain, more specifically, for example, the following formula (4):

(Wherein R ¹ represents a sugar residue)
A sugar chain having a double-stranded structure as represented by the following formula (5) or (6):

(Wherein R ¹ represents a sugar residue)
Or

(Wherein R ¹ represents a sugar residue)
A sugar chain having a three-chain structure represented by the following formula (7):

(Wherein R ¹ represents a sugar residue)
A sugar chain having a four-chain structure as represented by In the present invention, when a sugar chain is enzymatically synthesized, a sugar chain having a double-stranded structure can be preferably used from the viewpoint of ease of production.

  Examples of the sugar chain used in the present invention include a hybrid sugar chain having bisecting GlcNAc in addition to the above complex type sugar chain. Examples of the mixed sugar chain having bisecting GlcNAc used in the present invention include a sugar chain having a double chain structure, a sugar chain having a triple chain structure, and a sugar chain having a four chain structure.

Examples of the sugar residue represented as R ¹ in the sugar chain represented by the formulas (1) to (7) include a GlcNAc residue, a glucose residue, a fucose residue, a sialic acid residue, N- Examples include acetylgalactosamine residue, galactose residue, mannose residue, N-acetylmannosamine residue, xylose residue and the like.

R ² and R ³ in the sugar chain represented by the formula (2) represent a GlcNAc residue. R ² and R ³ may be bonded to a mannose residue via a β1-2 bond, β1-4 bond, or β1-6 bond, and in the sugar chain represented by the formula (2) May not exist. Further, when R ² is present in the sugar chain represented by the formula (2), one or two R ² can be bonded to one mannose residue. When two R ² are bonded to one mannose residue, the two R ² are bonded to the mannose residue in different binding modes. On the other hand, if R ³ is present in the sugar chain represented by the formula (2), said R ^3, like the above R ^2, for one mannose residue, one or two of R ³ Can be combined. In addition, when two R ³ are bonded to one mannose residue, the two R ³ are bonded to the mannose residue in a bonding manner different from each other in the same manner as R ² described above.

  Examples of the sugar chain having bisecting GlcNAc used in the present invention include sugar chains in which the non-reducing end is N-acetylglucosamine as represented by the above formulas (4) to (6). The present invention is not limited to these as long as the desired effect in the invention is exhibited. That is, the sugar chain having bisecting GlcNAc used in the present invention is a sugar chain in which the non-reducing end side of the sugar chain is further modified with galactose, sialic acid, fucose, glucuronic acid, GlcNAc, mannose, etc. Also good.

  In the present invention, the complex carbohydrate having a sugar chain having bisecting GlcNAc in its structure indicates a compound in which a sugar chain having bisecting GlcNAc is added to any protein, peptide, lipid, or the like. That is, examples of the complex carbohydrate having a sugar chain having bisecting GlcNAc used in the present invention include glycopeptides, glycoproteins, glycolipids and the like.

  As a method for producing a sugar chain having bisecting GlcNAc or a complex carbohydrate having the sugar chain in its structure (hereinafter referred to as “sugar chain or complex carbohydrate having bisecting GlcNAc”) used in the present invention, a desired method is used. There is no particular limitation as long as a sugar chain or a complex carbohydrate that exhibits the above effects is obtained. For example, a sugar chain or complex carbohydrate that does not have a bisecting GlcNAc obtained from a natural product is artificially bisecting GlcNAc. The method etc. which introduce | transduce are mentioned. The introduction of bisecting GlcNAc is not particularly limited, but can be performed enzymatically using N-acetylglucosaminyltransferase III (also referred to herein as “GnT-III”).

  Hereinafter, introduction of enzymatic bisecting GlcNAc by GnT-III will be described in detail. First, a sugar chain or a complex carbohydrate serving as a substrate for GnT-III is obtained.

  When glycoprotein is obtained as a complex carbohydrate used as the substrate, a known glycoprotein production method can be used. For example, calf serum fetuin, transferrin, γ-globulin and the like can be produced by known methods.

  In addition, when obtaining a glycopeptide as a complex carbohydrate used as the substrate, the glycoprotein is decomposed by limited protein hydrolysis using an enzyme such as trypsin, lysyl endopeptidase, chymotrypsin or the like. A glycopeptide can be obtained. When a glycoprotein is decomposed by protease-limited hydrolysis with a protease, an enzymatic reaction is carried out in a dialysis membrane or an ultrafiltration membrane, whereby the glycopeptide produced by decomposition with the enzyme can be easily separated. Furthermore, the desired glycopeptide can be purified from the decomposed peptide and glycopeptide mixture by purification means such as centrifugation, gel filtration, ion exchange chromatography, hydrophobic chromatography, and lectin affinity chromatography.

  In addition, free glycopeptides present in eggs of mammals such as eggs, birds and fish can also be used as the substrate. In this case, the target glycopeptide can be purified from the yolk portion by a purification means such as solvent extraction, centrifugation, gel filtration, ion exchange chromatography, hydrophobic chromatography, lectin affinity chromatography, and the like. An example of obtaining glycopeptides from eggs is described below. After adding an equal amount of water to chicken egg yolk, add 1/10 amount of phenol / water (9: 1) mixture and stir well. The resulting mixture is then subjected to centrifugation to recover the supernatant. After concentrating the obtained supernatant, gel filtration chromatography using Sephadex G-50 is performed, and the fractions that became positive in the color reaction of neutral sugars by the phenol-sulfuric acid method are collected. Thereby, a glycopeptide having a double-chain complex sugar chain can be obtained. The structure of the glycopeptide thus obtained (SEQ ID NO: 1) is shown in the following formula (8).

  In addition, when obtaining a sugar chain used as a substrate, a chemical method such as hydrazine degradation or glycopeptidase A (derived from almonds such as Seikagaku Corporation) from the above-mentioned glycoproteins such as calf serum fetuin, transferrin and γ-globulin. Co., Ltd.), glycopeptidase F (flavobacterium-derived, “PNGase”, for example, manufactured by Takara Bio Inc.) and the like can be used to release complex sugar chains (sugars). Protein Glycan Research Method, Biochemical Experimental Method 23, published by Academic Publishing Center, 1989).

  When GnT-III is allowed to act on the sugar chain or complex carbohydrate obtained as described above, sialic acid, galactose residues or fucose residues are usually removed from these non-reducing ends. A known method may be used to remove the sugar residue at the non-reducing end. For example, sialic acid can be removed in a dilute solution of an inorganic acid such as dilute hydrochloric acid or dilute sulfuric acid at 50 ° C. to 100 ° C. About 10 minutes to 2 hours, preferably by releasing sialic acid by heating in a 0.025-0.1N hydrochloric acid solution or 0.025-0.1N sulfuric acid solution at 80 ° C. for 1 hour. Can be. When sugar residues are removed by an enzymatic method, sialic acid residues, galactose residues, fucose residues, and the like are removed by enzymatic treatment with sialidase, galactosidase, fucosidase, and the like. As a result, a sugar chain or complex carbohydrate whose non-reducing end is GlcNAc, which is a suitable substrate for GnT-III, can be obtained.

  As the sialidase used herein, for example, an enzyme having a wide substrate specificity such as a sialidase derived from Arthrobacter ureafaciens or a sialidase derived from Clostridium perfringens is preferably used. In addition, as the galactosidase, for example, enzymes with wide substrate specificity such as β-galactosidase derived from peas and β-galactosidase derived from Aspergillus niger are preferably used. As the fucosidase, for example, α-fucosidase derived from Streptomyces sp. 142 strain, α-fucosidase derived from bovine kidney, and the like can be used.

  Bisecting GlcNAc is enzymatically introduced by allowing GnT-III to act together with UDP-GlcNAc on the sugar chain or complex carbohydrate whose non-reducing end is GlcNAc thus obtained. At this time, N-acetylglucosaminyltransferase IV (also referred to as “GnT-IV” in the present specification) and N-acetylglucosaminyltransferase V (also referred to as “GnT-V” in the present specification) are optionally provided. ) Together with UDP-GlcNAc, various sugar chains or complex carbohydrates having a three-chain structure or a four-chain structure can be obtained.

  As the GnT-III, GnT-IV and GnT-V, purified enzymes described in Japanese Patent No. 2789283, Japanese Patent Laid-Open No. 6-197756, International Publication No. 98/26053, etc. are used.

  Further, as GnT-III, GnT-IV and GnT-V, a nucleic acid (gene) encoding GnT-III, a nucleic acid (gene) encoding GnT-IV, and a nucleic acid (gene) encoding GnT-V, respectively. Cell lysates, crude enzyme solutions or purified enzymes prepared from transformed animal cells or the like can also be used. For example, an expression vector for animal cells containing a construct in which a GnT-III gene, a GnT-IV gene or a GnT-V gene is linked downstream of the actin promoter and having a neomycin resistance gene is used as a B16 melanoma cell, rat adrenal pheochromocytoma GnT-III, GnT-IV or GnT- is obtained by transfecting the derived PC12 cells and the like by electroporation, lipofectamine, etc., and screening the transformed cells using G418 resistance as an index. Cells expressing V are obtained. The obtained cells are sonicated, and the supernatant obtained by removing insolubles by centrifugation can be used as a crude enzyme solution. After synthesizing the sugar chain compound or derivative thereof used in the present invention by an enzyme reaction using the crude enzyme solution or the purified enzyme, from the obtained enzyme reaction solution, for example, trade name: Sephadex G15 (Amersham Separation and purification of sugar chain compounds or their derivatives from enzymes, sugar nucleotides, salts, etc. by gel filtration chromatography using Bioscience), separation using a cellulose cartridge (manufactured by Takara Bio Inc.) Can do.

  In the present invention, examples of the sugar chain or complex carbohydrate having bisecting GlcNAc include a sugar chain or complex carbohydrate whose non-reducing end is GlcNAc. Further, in the present invention, the sugar chain or complex carbohydrate having bisecting GlcNAc is suitably the sugar chain or complex sugar whose non-reducing terminal side is modified with fucose, galactose, sialic acid, glucuronic acid, GlcNAc, mannose, etc. It may be quality.

  Further, the sugar chain or complex carbohydrate having bisecting GlcNAc used in the present invention includes a sugar chain or complex carbohydrate having bisecting GlcNAc obtained from a natural product in addition to the above-mentioned enzyme reaction product. It is. Examples of a method for selectively obtaining a sugar chain or complex carbohydrate having bisecting GlcNAc include a method using a lectin that specifically binds to a sugar chain having bisecting GlcNAc, such as kidney bean lectin (E4PHA). The Further, in the present invention, a glycoprotein having bisecting GlcNAc obtained by a known method, for example, γ-glutamyltranspeptidase, γ-globulin, etc. may be used as it is, and obtained by appropriately performing the treatment. It may be used as a glycopeptide or a sugar chain. In the sugar chain and complex carbohydrate thus obtained, the sugar residue on the non-reducing end side is not particularly limited, and examples thereof include a GlcNAc residue, a galactose residue, a sialic acid residue, a fucose residue, Any sugar residue such as a mannose residue may be used.

  Moreover, the sugar chain or complex carbohydrate having bisecting GlcNAc used in the present invention may be a sugar chain or complex carbohydrate produced by synthesis.

  In the present invention, any sugar chain or complex carbohydrate derivative having the bisecting GlcNAc may be used as long as it exhibits a neurite outgrowth action. In the present invention, examples of the derivative include compounds obtained by modifying the sugar chain or complex carbohydrate within a range where the desired activity, that is, the neurite outgrowth action is not lost. Examples of the modification include, but are not limited to, the introduction of a non-natural sugar residue, the addition of a labeling compound or a ligand (biotin, histidine-tag, etc.), and immobilization on a carrier. The

  In the case of a sugar chain derivative, for example, the derivative can be obtained by binding the reducing end to a compound having an amino group by a reductive amination reaction. In the present invention, for example, an aliphatic amine, sphingosine, phosphatidylethanolamine, or the like may be bound via the reducing end of the sugar chain in order to impart hydrophobic properties to the sugar chain. In the case of derivatives of glycoconjugates (glycopeptides), for example, derivatives are obtained by binding fatty acids, biotin, etc. using the amino group or N-terminal amino group of the lysine residue of the peptide portion. be able to.

  In order to confirm the purity and structure of the sugar chain, complex carbohydrate, and derivatives thereof used in the present invention, in the case of a sugar chain, the reducing end is labeled with a fluorescent substance such as 2-aminopyridine or pyrenehydrazine, The obtained labeled product is analyzed by HPLC or the like. In the case of glycoconjugates, for example, glycopeptides, the peptide portion is labeled with a fluorescent substance such as dansyl chloride and FITC, and the obtained labeled product is analyzed by HPLC. The yield and purity can be confirmed by analyzing the above. Such a label is also included in the derivatives used in the present invention. Moreover, the structure of the sugar chain, complex carbohydrate, and derivatives thereof used in the present invention can be confirmed by performing LC-MS analysis or TOF-MS analysis to determine the molecular weight.

  The sugar chain, complex carbohydrate and derivatives thereof having bisecting GlcNAc used in the present invention can be in the form of a salt in order to improve stability and solubility in water. , Sodium salts, potassium salts, calcium salts, ammonium salts, chlorides, carbonates and the like. Examples of the salt of the sugar chain, the salt of complex carbohydrate, or the derivative thereof are for general desalting such as, for example, trade name: Dowex 50W, trade name: Dowex MR3 (both manufactured by Dow Chemical). Using an ion exchange resin, it can be obtained as a free sugar chain, complex carbohydrate, or a derivative thereof, or can be exchanged with other salts.

  The effect of inducing neurite outgrowth by the neurite outgrowth inducer of the present invention is, for example, a nervous system cell, preferably, for example, neuroblastoma cell, chromaffin cell tumor cell, glial cell tumor cell, etc. In the presence of a cell having a neurite, the sample has an indicator that the sample has a neurite outgrowth-inducing effect, and is evaluated by observing the obtained cell. Become. Moreover, the effect | action in cell extension can also be evaluated by the same method.

  Specific examples of the neural cells include SK-N-SH cells that are human neuroblastoma cell lines such as Neuro-2a cells (ATCC CCL-131) that are mouse neuroblastoma cell lines. (ATCC HTB-11), SH-SY5Y cells, rat pheochromocytoma cell line PC12 cells (ATCC CRL1721) and the like, rat astroglioma cell line C6 cells (ATCC CCL-107) and the like. It is done.

The culture conditions for the nerve cells are not particularly limited, but 0.01% by weight poly-L-lysine-coated culture dish in Dulbecco's modified Eagle medium (DMEM) containing 10% by weight horse serum and 5% by weight FCS. Inoculated to 1 × 10 ⁵ cells / dish and cultured at 37 ° C. in the presence of 5% by volume CO ₂ .

  Moreover, the density | concentration of the sample of the test subject in a culture medium can be set suitably.

  Formation of neurites in cells can be observed, for example, with a phase contrast microscope. In the present invention, for example, preferably, cells having two or more neurites of 3 μm or more are “cells having neurites”, and cells having low brightness when viewed with a phase contrast microscope are “stretched cells”. It is evaluated as.

  Moreover, you may evaluate the differentiation-inducing ability with respect to the cell about the neurite extension inducer of this invention. The differentiation induction ability is determined by culturing the nervous system cells in the presence of a sample to be tested, and regarding the obtained cells, whether or not various markers serving as indicators of cell differentiation, such as neurofilament H, are expressed. It can be evaluated by measuring.

  The sugar chain, complex carbohydrate, derivative thereof and / or salt thereof (hereinafter sometimes referred to as the active ingredient of the present invention) having bisecting GlcNAc used in the present invention has neurite outgrowth activity, It can be used as a neurite outgrowth agent (that is, a neurite outgrowth inducing agent), and thus as a therapeutic agent or prophylactic agent effective in the treatment of neurological diseases, and an agent for improving or improving learning and / or memory ability. The neurological disease means a disease accompanied by neurodegeneration, for example, a brain tumor (for example, a brain tumor with nerve damage or a decrease in nerve function); Alzheimer's disease, Parkinson's disease, Huntington's chorea, Creutzfeldt-Jakob disease, muscle atrophy Examples include neurodegenerative diseases such as lateral sclerosis; dementia; diseases such as cerebral ischemic injury, and the like.

  Furthermore, in the present invention, an enzyme related to the synthesis of a sugar chain having the bisecting GlcNAc and a nucleic acid encoding the enzyme can also be used as the active ingredient. Although it does not specifically limit as said enzyme, GnT-III etc. are illustrated. Examples of the nucleic acid encoding GnT-III include a rat-derived nucleic acid encoding GnT-III (Journal of Biological Chemistry, Vol. 267, pages 18199 to 18204, 1992), human-derived GnT-III. And the nucleic acid to be encoded [Journal of Biochemistry, Vol. 113, pages 692 to 698, 1992].

  According to the GnT-III, a sugar chain having a bisecting GlcNAc is synthesized in a cell to which GnT-III is administered, thereby exhibiting an excellent effect of expressing a neurite outgrowth action. In addition, according to the nucleic acid encoding GnT-III, in the cell to which the nucleic acid encoding GnT-III is administered, the nucleic acid is translated from the nucleic acid into GnT-III, and has a bisecting GlcNAc by the GnT-III. A chain is synthesized, thereby exhibiting an excellent effect of developing a neurite extension action.

  Therefore, in another aspect, the present invention relates to an agent for treating or preventing neurological diseases, an agent for treating or preventing dementia, an agent for improving or improving learning and / or memory ability.

  In addition, in another aspect, the present invention provides the bisecting for the production of a therapeutic agent or preventive agent for neurological diseases, a therapeutic agent or preventive agent for dementia, an agent for improving or improving learning ability, and the like. Sugar chain having GlcNAc, in particular, the sugar chain represented by the above formula (2), complex carbohydrate having the sugar chain in the structure, derivative of the sugar chain, derivative of the complex carbohydrate, drug of the sugar chain Physiologically acceptable salt, pharmacologically acceptable salt of the glycoconjugate, at least one use selected from the group consisting of an enzyme involved in the synthesis of the sugar chain and a nucleic acid encoding the enzyme .

  Since the neurite can be elongated by the sugar chain having the bisecting GlcNAc, glycoconjugate, derivative thereof and / or salt thereof, treatment of a neurological disease in an individual, for example, a human, a non-human animal or the like Prevention, learning and / or improvement or improvement of memory ability are possible.

  The therapeutic agent or preventive agent for neurological diseases, the therapeutic agent or preventive agent for dementia, the improving agent or improving agent for learning and / or memory ability according to the present invention is a commonly used preparation form such as an injection, an oral agent, an ointment, etc. However, it is preferably administered directly to the affected area as an injection. When dispensing as an injection, a buffer solution such as PBS, a pH adjusting agent, physiological saline, a stabilizer and the like are added to the sugar chain derivative of the present invention, and intravenous, intradermal, subcutaneous, muscle is added by a conventional method. And injection for intravenous administration can be prepared.

  The neurite outgrowth inducer, the therapeutic or preventive agent for neurological diseases, the learning and / or memory ability improving agent of the present invention is a sugar chain having bisecting GlcNAc, and a complex sugar having the sugar chain in its structure A substance containing at least one selected from the group consisting of a saccharide, a derivative of the sugar chain or a complex carbohydrate, and a pharmacologically acceptable salt of the sugar chain or the complex carbohydrate The dose varies depending on the patient (for example, body weight, age, medical history, etc.) and the indication case. For example, the injection may be administered so that the amount of the active ingredient is about 0.001 to 100 mg per injection. preferable.

  When the neurite outgrowth-inducing agent, therapeutic agent or preventive agent for neurological diseases of the present invention, or an agent for improving or improving learning and / or memory ability contains GnT-III as an active ingredient, administration to cells Can be performed by a method used for a known protein pharmaceutical, for example, by administering it as an injection to a target cell or its surrounding tissue.

  When the neurite outgrowth inducer, the therapeutic or preventive agent for neurological diseases, the improving or improving agent for learning and / or memory ability of the present invention contains a nucleic acid encoding GnT-III as an active ingredient, Administration to cells can be performed by known gene transfer methods.

  Examples of the gene introduction method include calcium phosphate method, microinjection method; physicochemical method such as electroporation method, lipofection method, liposome method; adenovirus, SV40 virus, herpes simplex virus, adeno-associated virus, retrovirus. And a method using a viral vector such as a lentivirus. In the physicochemical method, a DNA fragment containing the nucleic acid or a vector into which the nucleic acid has been inserted, for example, a plasmid vector, is introduced into a cell. When a continuous effect is desired, it is preferable to use a viral vector having a property of integrating a target gene into a chromosome, such as adeno-associated virus, retrovirus, and lentivirus.

  In addition, the neurite outgrowth agent of the present invention to the living body, brain tumor / neurodegenerative disease / dementia, therapeutic agent or preventive agent for cancer / malignant tumor, learning and / or memory ability improver or improver are active ingredients As a nucleic acid incorporated into a DNA fragment containing a nucleic acid or an appropriate vector, it may be administered to a site where administration is desired, or the cell into which the nucleic acid has been introduced may be transplanted to a site where administration is desired .

  If GnT-III or a nucleic acid encoding GnT-III is administered to a cell in the vicinity of a cell (target cell) where neurite outgrowth is desired (target cell), even if it is not administered to the target cell, Since the sugar chain having bisecting GlcNAc produced in cells exerts neurite outgrowth action on target cells, the object of the present invention can be achieved.

  The therapeutic or prophylactic agent for neurological diseases of the present invention comprises a sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, the sugar chain or a derivative of the complex carbohydrate, the sugar chain or the complex sugar One major feature is that it contains as an active ingredient at least one selected from the group consisting of a quality pharmacologically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. . Therefore, according to the therapeutic agent or preventive agent for neurological diseases of the present invention, it exhibits an excellent effect of inducing neurite outgrowth in an individual suffering from a neurological disease, thereby treating or preventing the neurological disease. To do.

  The pharmacological evaluation of the therapeutic or prophylactic agent for neurological diseases of the present invention is performed by, for example, an in vitro evaluation method by the same method as the evaluation of the neurite outgrowth induction effect; an evaluation method using a neurological disease model animal; Analysis by positron emission tomography in an affected individual; measurement of brain function by brain magnetic field measurement in an individual suffering from neurological disease; functional analysis by drawing magnetic resonance function in an individual suffering from neurological disease. The evaluation method using a neurological disease model animal includes, for example, administering a drug to be evaluated to a model animal such as a mouse, rat, rabbit, dog, monkey, etc., and changing a physiological index based on the disease in the model animal and / or It is performed by diagnosing changes in biochemical indicators, observing diseased sites by anatomical examination, and the like. Here, in the model animal, the physiological index and / or the biochemical index is a physiological index in a normal animal that is a source of a neurological disease model animal (that is, an individual substantially not suffering from a neurological disease) and When the state similar to the biochemical index is indicated, the disease state in the diseased part is the same as the corresponding part in the normal animal, etc., the drug to be evaluated is treatment or prevention of neurological disease It becomes an index of having an effect. In addition, analysis by positron emission tomography is performed at a disease site, for example, a neurotransmitter such as dopamine, serotonin, histamine, noradrenaline, acetylcholine, opiate, γ-aminobutyric acid (GABA), glutamate, etc. This can be done by analyzing Here, when the turnover or the like shows a state similar to the corresponding site in a normal individual, it becomes an index that the drug to be evaluated has a therapeutic or preventive effect on a neurological disease.

  The learning and / or memory ability improver or improver of the present invention includes a sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, the sugar chain or a derivative of the complex carbohydrate, the sugar 1 containing at least one selected from the group consisting of a chain or a pharmacologically acceptable salt of the complex carbohydrate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme as an active ingredient There are two major features. Therefore, the agent for improving and / or improving learning and / or memory ability of the present invention induces neurite outgrowth in an individual in need of improvement or improvement of learning and / or memory ability, thereby / Or memory capacity can be improved or improved.

  In the present specification, “improvement of learning and / or memory ability” means substantially the level of learning and / or memory ability that is normally exhibited by normal individuals such as humans, non-human animals, etc. Means to increase. In addition, “improvement of learning and / or memory ability” means an individual who has substantially lost learning and / or memory ability due to neurological disease, cerebral ischemic injury, trauma, etc. It means to substantially increase the level of learning and / or memory ability in an individual.

  The pharmacological evaluation of the learning and / or memory ability improving agent or improving agent of the present invention includes, for example, an evaluation method using a normal animal, a model animal having low learning and / or memory ability, a learning ability test, a memory ability test, etc. It can be done by. The evaluation method using the normal animals, model animals, etc. is, for example, the time to escape from the maze where the drug to be tested is administered to animals such as mice, rats, rabbits, dogs, monkeys, and food is placed at the goal point. This can be done by measuring several times. Here, compared to a normal animal to which the test subject drug is not administered, in the normal animal to which the test subject drug is administered, the time required for the time from the start point to the goal point where the food is placed is larger, etc. It is an indicator that the drug to be tested shows improved learning and / or memory ability. In addition, compared to the model animal before administering the test subject drug, in the model animal administered the test subject drug, the time required from the start point to the goal point where the food was placed was further shortened, etc. This is an indicator that the drug to be tested shows improved learning and / or memory ability.

  In another aspect, the present invention relates to a method for inducing neurite outgrowth.

  The method for inducing neurite outgrowth according to the present invention comprises a sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, and a pharmacology of the sugar chain At least one selected from the group consisting of pharmaceutically acceptable salts, pharmacologically acceptable salts of glycoconjugates, enzymes involved in the synthesis of the sugar chains, and nucleic acids encoding the enzymes One feature is to do this.

  Examples of the specimen include cultured cells, nerve tissues, and individual animals. Examples of the animal individual include humans and non-human animals.

  The effect of inducing neurite outgrowth by the induction method of the present invention can be evaluated by the same method as the evaluation of the neurite outgrowth induction effect by the neurite outgrowth inducer.

  In the induction method of the present invention, administration can be performed by the same method as in the case of the therapeutic agent or preventive agent for neurological diseases. In the induction method of the present invention, the neurite outgrowth inducer of the present invention may be used.

  Cultured cells (eg, nervous system cells, neural progenitor cells) and neural tissue in which neurites have been elongated by the induction method of the present invention can be used for transplantation for the purpose of treating neurological diseases.

  In another aspect, the present invention relates to a method for treating or preventing a neurological disease.

  The method for treating or preventing a neurological disease of the present invention is a sugar chain having the bisecting GlcNAc, particularly represented by the formula (2), in an individual suffering from a neurological disease or an individual who is likely to suffer from the neurological disease. A sugar chain having a bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, One feature is administration of at least one selected from the group consisting of a pharmacologically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme.

  Examples of the individual include humans and non-human animals.

  The therapeutic or prophylactic effect of the neurological disease treatment or prevention method of the present invention can be evaluated by the same method as the pharmacological evaluation of the neurological disease therapeutic agent or prophylactic agent of the present invention.

  In addition, in the method for treating or preventing a neurological disease of the present invention, administration can be performed by the same method as that for the therapeutic agent or preventive agent for the neurological disease of the present invention. In the method for treating or preventing a neurological disease of the present invention, the therapeutic agent or preventive agent for a neurological disease of the present invention may be used.

  In still another aspect, the present invention relates to a method for improving or improving learning and / or memory ability.

  According to the method for improving or improving learning and / or memory ability of the present invention, a sugar chain having the bisecting GlcNAc, particularly a bisecte represented by the formula (2), is applied to an individual who needs to improve or improve learning ability. A sugar chain having a ting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, One major feature is to supply at least one selected from the group consisting of a pharmacologically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme.

  Individuals who need to improve or improve the learning ability include normal individuals such as humans and non-human animals; learning and / or memory ability is substantially lost due to neurological diseases, cerebral ischemic injury, trauma, etc. An individual whose learning and / or memory ability has decreased due to neurological disease, cerebral ischemic injury, trauma, or the like.

  The improvement or improvement effect of learning and / or memory ability by the method for improving or improving learning and / or memory ability of the present invention is evaluated by the same method as the pharmacological evaluation of the improver or improver of the learning and / or memory ability. Can be done.

  Moreover, in the method for improving or improving learning and / or memory ability of the present invention, administration can be performed by the same method as in the therapeutic agent or preventive agent for neurological disease of the present invention. In the method for improving or improving the learning and / or memory ability of the present invention, the agent for improving or improving the learning and / or memory ability of the present invention can be used.

  In another aspect, the present invention relates to a food or beverage.

  The food or beverage of the present invention comprises a sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, and a pharmacologically acceptable sugar chain. One major characteristic is that it contains at least one selected from the group consisting of a salt that can be prepared and a pharmacologically acceptable salt of the complex carbohydrate (the active ingredient). Therefore, the food or beverage of the present invention is extremely useful for symptom improvement and prevention of the above diseases due to its neurite outgrowth action. Furthermore, the food or beverage may be a functional food or a functional beverage (for example, a food for specified health use) that indicates that the purpose is to improve or prevent the symptoms of the disease.

  Since the food or beverage of the present invention contains the active ingredient, it can be used for the treatment or prevention of neurodegenerative diseases, dementia or brain tumors. Furthermore, since the food or drink of the present invention contains the active ingredient, it can be used for improving or preventing learning and / or memory ability. The food or beverage of the present invention includes a food or beverage for assisting in the treatment or prevention of neurodegenerative diseases, dementia or brain tumors, and a food or beverage for assisting in the improvement or prevention of learning and / or memory ability. Beverages are also included.

  Here, the term “containing” used for the food or beverage of the present invention includes the meanings of “addition” and / or “dilution”.

  In the present specification, the “containing” used for the food or beverage of the present invention is an aspect in which the active ingredient used in the present invention is contained in the food or beverage, and the “addition” The aspect of adding the active ingredient used in the present invention to the raw material of food or beverage, and the “dilution” is the aspect of adding the raw material of food or beverage to the active ingredient used in the present invention. Say.

  It does not specifically limit to the manufacturing method of the foodstuff or drink of this invention. For example, blending, cooking, processing, and the like may be performed in accordance with general food or beverage production methods, and can be produced by those production methods, and the obtained food or beverage may contain the active ingredient. .

  The food or beverage of the present invention is not particularly limited as long as it contains the above-mentioned active ingredient. For example, processed food products (processed flour products, processed starch products, premix processed products, noodles, Macaroni, bread, sweet potato, buckwheat, rice bran, rice noodles, harsame, packaging rice cake), processed oils and fats (plastic oil, tempura oil, salad oil, mayonnaise, dressing, etc.), processed soybean products (tofu, miso , Natto, etc.), processed meat products (ham, bacon, press ham, sausage, etc.), marine products (frozen surimi, kamaboko, chikuwa, hanpen, fried fish, tsumire, streaks, fish ham, sausage, bonito, fish egg processed products, Canned seafood, boiled Tsukuda, etc.), dairy products (raw milk, cream, yogurt, butter, cheese, condensed milk, powdered milk, ice cream, etc.), processed vegetables and fruits (pae) Tomatoes, jams, pickles, fruit drinks, vegetable drinks, mixed drinks, etc.), confectionery (gum, candy, chocolate, biscuits, confectionery breads, cakes, candy sweets, rice confectionery, etc.), alcoholic drinks (sake, Chinese liquor, wine, whiskey, shochu, vodka, brandy, gin, rum, beer, soft alcoholic beverage, fruit liquor, liqueur, etc.), beverages (green tea, tea, oolong tea, coffee, soft drink, lactic acid beverage, etc.), Seasonings (soy sauce, sauce, vinegar, mirin, etc.), canned / bottled / bagged foods (beef rice, kettle rice, red rice, curry, other cooked foods), semi-dried or concentrated foods (lever paste, other spreads) , Buckwheat noodle soup, concentrated soup), dried food (instant noodles, instant curry, instant coffee, powdered juice, powdered soup, instant miso , Cooked foods, cooked beverages, cooked soups, etc.), frozen foods (sukiyaki, steamed tea bowls, eel kabayaki, hamburg steak, shumai, dumplings, various sticks, fruit cocktails, etc.), solid foods, liquid foods (soups, etc.) Agricultural and forestry processed products such as spices, livestock processed products, and processed fishery products.

  The food or beverage of the present invention contains the above-mentioned active ingredient, alone or in combination, mixed, added and / or diluted, as long as it contains a necessary amount for developing a neurite outgrowth action. Often, it includes forms that can be taken orally, such as tablets, granules, and capsules.

  The content of the active ingredient in the food or beverage of the present invention can be appropriately selected from the viewpoint of its function and neurite outgrowth activity. For example, in the food or beverage of the present invention, 0.01% by weight or more, preferably 0.1% by weight or more, more preferably 1% by weight or more, 10% by weight or less, more preferably 5% by weight or less. The content can be arbitrarily selected within the above range. The food or beverage of the present invention is preferably such that the active ingredient contained therein is 0.00001 mg to 1000 mg / kg body weight, preferably 0.0001 mg to 100 mg / kg body weight per day for a human (for example, an adult). Preferably, it may be taken so as to obtain an intake of 0.001 mg to 10 mg / kg body weight. Such intake may vary depending on various conditions, and an amount smaller than the above intake may be sufficient or may be required beyond the range.

  Evaluation of the food or beverage of the present invention can be carried out, for example, by an evaluation method using a neurological disease model animal used for pharmacological evaluation of the therapeutic agent or prophylactic agent of the neurological disease of the present invention.

  By the way, a sugar chain produced by the action of GnT-V and having a GlcNAc residue at the non-reducing end and having a three-chain structure or a four-chain structure and a complex carbohydrate having the sugar chain are as follows: The therapeutic agent or metastasis inhibition effective for the treatment of malignant tumors by inhibiting the invasion or metastasis of cancer cells because it has the activity of inhibiting cell migration and movement as apparent from the suppression of wound closure described in the examples It can be used as an agent. The therapeutic agent or metastasis inhibitor for malignant tumor can be produced according to the therapeutic agent or prophylactic agent for neurological diseases of the present invention.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to such examples.

Production of Gn (Gn) Gn-bi-glycopeptide (1) Preparation of sialylglycopeptide (SGP) from chicken egg yolk An equal amount of water was added to 1 L (50 pieces) of white leghorn fresh egg yolk. To the obtained egg yolk dilution, 1/10 amount of phenol / water (9: 1, weight ratio) was added and stirred vigorously for 2 hours. Centrifugation was performed at 7000 × g (6000 rpm) for 30 minutes to obtain a supernatant. The supernatant was concentrated to 6 mL with a rotary evaporator to remove unnecessary substances. The obtained product was subjected to gel filtration using a trade name: Sephadex G-50 (Amersham Biosciences) column (2.5 cm × 100 cm). At this time, 0.1 M NaCl was used as the eluent. The sugar contained in the eluted fractions was converted to the phenol-sulfuric acid method (edited by the Japanese Biochemical Society, New Chemistry Laboratory, Volume 3, Carbohydrate I, Glycoprotein (above), page 143, 1990, published by Tokyo Chemical Doujin. ) And the second major eluted saccharide positive fraction was collected. Using a rotary evaporator, the sugar positive fraction was concentrated to 5 mL. The obtained product was desalted using a Sephadex G-25 (Amersham Biosciences) column (2.5 cm × 52 cm). At this time, 5 volume% ethanol was used as an eluent. The sugar content of each fraction was measured by the phenol sulfuric acid method, and sugar-positive fractions were collected. The obtained product was concentrated to 5 mL with a rotary evaporator to obtain a concentrated solution. Product name: TOYOPEARL DEAE-650M (manufactured by Tosoh Corporation, product number: 07473) equilibrated with 5 mM Tris-HCl (pH 8.0) was loaded onto the column (3.0 cm × 24 cm) with the concentrated solution, and the fraction passed through. Collected. The flow-through fraction was concentrated to 5 mL using a rotary evaporator. The obtained product was desalted using a trade name: Sephadex G-25 column and freeze-dried to obtain SGP.

(2) Removal of sialic acid and galactose from SGP by enzyme treatment SGP obtained in Example 1- (1) was dissolved in water so as to be 170 μmol / 1.5 mL. 45 mg of powder EZ-Link Sulfo-NHS-Biotin (trade name, manufactured by PIERCE, product number: 21217) was added to the obtained solution, and the mixture was rapidly stirred vigorously and then shaken at room temperature for 1 hour. The resulting mixture was concentrated to 0.5 mL using a centrifugal concentrator. To the obtained concentrate, 2.4 mL of 100 mM sodium citrate buffer (pH 5.0) and 3 U / 300 μL of neuraminidase (manufactured by Nacalai Tesque, product number: 24229-74) were added and reacted at 37 ° C. for 24 hours. I let you. 3U / 70 μL of β-galactosidase (manufactured by Seikagaku Corporation, product number: 100570) was added to the obtained product, and the mixture was further reacted for 24 hours. After the reaction, the obtained reaction product was heated at 98 ° C. for 5 minutes to inactivate the enzyme, and centrifuged at 13000 × g (14000 rpm) for 10 minutes. The obtained supernatant was concentrated to 400 μL using a centrifugal concentrator. The obtained product was subjected to a product name: PD-10 desalting column (manufactured by Amersham Biosciences, product number: 17-0851-1) for desalting, and further a cellulose cartridge column (manufactured by Takara Bio Inc.). , Product number: 4404) and purified to obtain a crude purified biotinylated Gn.Gn-bi-glycopeptide (hereinafter, “crude purified Gn.Gn-bi-GP”). The structure of Gn.Gn-bi-GP (SEQ ID NO: 1) is shown in the following formula (9).

(3) Introduction of bisecting GlcNAc residue by GnT-III Rat GnT-III cDNA [Nishikawa, A. et al., Journal of Biological Chemistry, 267, 18199-18204, 1992] Plasmid pCXNII / GnT-III was constructed by subcloning into the EcoRI site of pCXNII [Niwa et al., Gene, 15, 193-199, 1991].

Using the product name: Lipofectamine 2000 (manufactured by Invitrogen, product number: 11668-019), the rat adrenal pheochromocytoma cell line PC12 (ATCC CRL-1721) was transfected with the pCXNII / GnT-III, The cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% by weight horse serum, 5% by weight fetal calf serum (FCS), and 1 mg / mL G418 at 37% at 5% CO ₂ . Colonies were isolated after 2 weeks of culture and recloned by limiting dilution to obtain PC12 (GnT-III) cells. In the same manner, PC12 cells were transfected with the pCXNII and cloned to obtain PC12 (mock) cells. The obtained PC12 (GnT-III) cells were DMEM containing 10% by weight horse serum and 5% by weight FCS on three collagen-coated 10 cm dishes (manufactured by Asahi Techno Glass, product number: 4020-010). Incubated in the presence of 5% by volume CO ₂ at 37 ° C. until confluent.

  After completion of the culture, the medium was discarded and washed with phosphate buffered saline [PBS (−)]. The cells were detached from the dish using a cell scraper and centrifuged at 1500 × g (5000 rpm) for 5 minutes to collect the cells. The supernatant was discarded, and 200 μL of PBS (−) containing 10 μg / mL leupeptin, 10 μg / mL aprotinin and 1 mM phenylmethylsulfonyl fluoride was added to the obtained cells, and the cells were suspended by pipetting. This cell suspension was subjected to sonication, and the obtained product was centrifuged at 700 × g for 10 minutes to obtain about 200 μL of the supernatant. The obtained supernatant was used as a GnT-III crude enzyme solution.

100 μL of GnT-III crude enzyme solution and 200 μL of 2 × buffer [Composition: 250 mM MES-NaOH (pH 6.25), 20 mM MnCl ₂ , 400 mM N-acetylglucosamine (GlcNAc), 1.0% (V / V ) Triton ^™ X-100], 40 μL of 500 mM UDP-GlcNAc, 50 μL of 0.825 μmol / 50 μL crude purified Gn.Gn-bi-GP aqueous solution prepared in Example 1- (2), 10 μL of water Were mixed and reacted at 37 ° C. for 24 hours. After the reaction, the enzyme was inactivated by heating at 98 ° C. for 5 minutes, and centrifuged at 13000 × g (14000 rpm) for 10 minutes to obtain a supernatant. Product name: The supernatant was desalted using a PD-10 column, purified using a cellulose cartridge column, concentrated under reduced pressure, and dried. The obtained product was dissolved in 200 μL of water to obtain a sample A containing a crude purified Gn (Gn) Gn-bi-glycopeptide. The glycopeptide concentration in sample A was measured by the phenol sulfate method and found to be 4.1 mM. The structure of Gn (Gn) Gn-bi-glycopeptide (SEQ ID NO: 1) is shown in the following formula (10).

(4) Sugar chain structure analysis of sample A The sugar chain structure of sample A was analyzed by the following method. Sample A diluted 100-fold with water 1 μL, 0.5 μL of 0.5 M sodium phosphate buffer (pH 7.5), 0.5 μL of 10 vol% Nonidet (trade name) P / 40 (NP- 40, manufactured by Merck, product number: BDH560092L), 0.5 μL of 500 U / μL peptide: N-glycosidase F (PNGase F, manufactured by New England BioLabs, product number: P0704S); 5 μL of water was added and reacted at 37 ° C. for 24 hours. The obtained reaction solution was boiled for 5 minutes, and a supernatant was obtained by centrifugation at 13000 × g (14000 rpm) for 5 minutes.

The whole amount of the supernatant was converted to pyridylamino (PA) using a trade name: GlycoTAG Reagent Kit (manufactured by Takara Bio Inc., trade number: GT500). The PA-converted sugar chain was dissolved in 45 μL of water, and 20 μL of the sugar chain was analyzed by HPLC. In the HPLC, as a column, trade _{name: TSKgel ODS-80T M (4.6} × 150mm, manufactured by Tosoh Corporation, product number: 08148), as eluent, 0.2% by volume of 1-butanol containing 20mM ammonium acetate buffer The liquid (pH 4.0) was used, the column temperature was set to 50 ° C., and the flow rate was set to 1.0 ml / min. A fluorescence detector was used for detection, and the excitation wavelength was set to 320 nm and the detection wavelength was set to 400 nm. Under these conditions, the PA-Gn.Gn-bi-sugar chain was eluted at 7.1 minutes and the PA-Gn (Gn) Gn-bi-sugar chain was eluted at 13.0 minutes.

  As a result, PA-Gn (Gn) Gn-bi-glycan was not detected from the crude purified Gn.Gn-bi-GP, whereas 15% PA-Gn (Gn) Gn was detected from the sample A. -bi-sugar chain was detected.

Production of Gn.Gn.Gn-tri'-glycopeptide (1) Introduction of β1,6-GlcNAc residue into biotinylated Gn.Gn-bi-glycopeptide Gu et al. [Gu, J. et al. , Journal of Biochemistry, Vol. 113, pp. 614-619, 1993], GnT-V was purified from human small cell lung cancer cell line QG. Using the obtained GnT-V, a reaction similar to Example 1- (3) was performed on the crude purified Gn.Gn-bi-GP to obtain Sample B. In the reaction using GnT-V, 2 × buffer solution [composition: 250 mM MES-NaOH (pH 6.25), 400 mM GlcNAc, 20 mM EDTA · 3Na, 1.0 vol% Triton ^™ X- 100] was used.

  The structure of the product Gn.Gn.Gn-tri′-glycopeptide (SEQ ID NO: 1) is shown in the following formula (11).

(2) Sugar chain structure analysis of sample B Sample B was analyzed by PNGase F treatment, PA conversion, and HPLC under the same conditions as in Example 1- (3). Under these conditions, PA-Gn.Gn-bi-sugar chains were eluted at 7.1 minutes and PA-Gn.Gn.Gn-tri'-sugar chains were eluted at 5.2 minutes.

  As a result, PA-Gn.Gn.Gn-tri'-sugar chain was not detected from the crude purified Gn.Gn-bi-GP, whereas 63% PA-Gn.Gn. Gn-tri'-sugar chain was detected.

Neurite formation and cell extension by Gn (Gn) Gn-bi-glycopeptide and Gn.Gn.Gn-tri'-glycopeptide (1) Gn (Gn) Gn-bi-glycopeptide and Gn.Gn.Gn-tri Neurite formation and cell expansion of Neuro-2a cells by '-glycopeptide 200 μL of the GnT-III crude enzyme solution prepared by the method of Example 1- (3) was heat-treated at 98 ° C. for 5 minutes, and 13000 × g ( The supernatant was obtained by centrifugation at 14,000 rpm for 10 minutes. The obtained supernatant was loaded onto a cellulose cartridge column (manufactured by Takara Bio Inc., product number: 4404) to obtain an elution fraction. This fraction was concentrated to dryness by centrifugation and dissolved in 200 μL of water to obtain Sample C.

  Sample D was obtained from PC12 (mock) cells by the same method.

The mouse neuroblastoma cell line Neuro-2a (ATCC CCL-131) was suspended in DMEM containing 10% by weight horse serum and 5% by weight FCS and 0.01% poly-L-lysine [Sigma (SIGMA). ), Product number: P4832] 35 mm glass bottom culture dish [MatTek, product number: P35G-0-10-C] inoculated to 1 × 10 ⁵ pieces / dish And cultured overnight at 37 ° C. in the presence of 5% by volume CO ₂ .

  After removing the culture medium by suction, 100 μL of a diluted sample obtained by diluting sample A, sample C or sample D 100-fold with PBS (−) containing 10 mM HEPES is added to the dished portion and incubated at 37 ° C. for 90 minutes. did. Thereafter, the cells were fixed with PBS (-) containing 3.7% by volume formalin. The fixed cells were observed with a phase contrast microscope (manufactured by Carl Zeiss, trade name: Axiovert 200M), and the ratio of cells having neurites and the ratio of expanded cells were calculated. Here, a cell having two or more neurites of 3 μm or more is referred to as “cell having neurite”, and a cell having low brightness when observed with a phase contrast microscope is referred to as “stretched cell”.

  The results are shown in FIG. FIG. 1 is a graph showing the ratio of the number of cells having neurites and the number of expanded cells to the total number of cells, and shows the average and standard deviation of three fields of view. In the sample A addition group, most cells showed neurite outgrowth and cell extension. On the other hand, although neurite formation was slightly observed in the sample C addition group, cell extension was not observed, and in the sample D addition group, neurite formation and cell extension were hardly observed.

  As the test sample, crude purified Gn.Gn-bi-GP, sample A and sample B were used, and the cells were incubated in the same manner as described above, and formalin fixation was performed 90 minutes after addition of the test sample. After formalin fixation, the plate was washed with PBS (−). Subsequently, actin was stained with Alexa 546-phalloidin, and observation with a confocal laser microscope (manufactured by Carl Zeiss, product number: LSM 5 Pascal) was also performed.

  As a result, most of the cells were spherical and almost no neurites were seen in the roughly purified Gn.Gn-bi-GP addition group, whereas the sample A addition group containing Gn (Gn) Gn-bi-GP was observed. The majority of cells formed numerous neurites and showed cell extension. In addition, in the sample B addition group containing Gn.Gn.Gn-tri'-GP, most of the cells were spherical and almost no neurite formation was observed, but the actin cytoskeleton reorganization like a lamellipodia was seen. It was.

(2) Induction of differentiation of Neuro-2a cells by Gn (Gn) Gn-bi-glycopeptide Neuro-2a cells were cultured in the same manner as in Example 3- (1), and then the medium was removed and 100 times with DMEM. Diluted sample A, DMEM, or 2 mL of DMEM containing 10 wt% FCS was added and cultured at 37 ° C. for 36 hours in the presence of 5 vol% CO ₂ . The medium was removed and the cells were fixed with PBS containing 4% by weight paraformaldehyde for 1.5 minutes. Subsequently, the mixture was gently shaken in 50 mM Tris-HCl (pH 6.8) containing 1 wt% Triton ^™ X-100 at room temperature for 15 minutes. Cells were re-fixed with 4 wt% paraformaldehyde for 30 minutes at 4 ° C. Cells were washed with PBS and incubated for 30 minutes in 50 mM NH ₄ Cl. The cells were then anti-phosphorylated neurofilament H (NF-H) mouse monoclonal antibody SMI-31 diluted 2000 times with PBS containing 2% by weight bovine serum albumin and 0.1% by weight Triton ^™ X-100. Incubated overnight at 4 ° C. in Sternberger Monoclonals, product number: SMI-31. Further, the obtained cells were incubated at room temperature for 30 minutes in PBS containing 10% by weight goat serum, and incubated for 2 hours at room temperature in Alexa 546-labeled goat anti-mouse IgG diluted 300-fold with PBS. In addition, it wash | cleaned 2-3 times with PBS for every incubation. The cells after incubation were observed with a confocal laser microscope.

  As a result, almost no neurite formation was observed in the DMEM-added group containing 10 wt% FCS, and NF-H positive neurites were observed in some cells in the DMEM-added group. More NF-H positive neurites were observed in the sample A addition group than in the DMEM addition group.

(3) Extension of T98G cells by Gn (Gn) Gn-bi-glycopeptide Experiments similar to Example 3- (1) were performed using human glioma cells T98G (ATCC CRL-1690). However, roughly purified Gn.Gn-bi-GP, sample A and sample B were used as test samples, and dilution with PBS (-) containing 10 mM HEPES was performed 50 times. The time from sample addition to formalin fixation was 90 minutes, and actin was observed by phase contrast microscopy and Alexa 546-phalloidin (manufactured by Invitrogen).

  As a result, in the group with the addition of the crude purified Gn.Gn-bi-GP and the group with the sample B, the cell extension was inhibited and spherical, whereas in the group with the sample A added, most of the cells extended, and the stress fiber Formed remarkably.

Inhibition of T98G motility by Gn.Gn.Gn-tri'-glycopeptide 0.5 × 10 ⁶ T98G cells per well were seeded in a 24-well plate, 5 volumes in RPMI 1640 medium containing 10 wt% FCS The cells were cultured overnight at 37 ° C. in the presence of% CO ₂ . Using a blue chip, a line was drawn at the center of the well to make a wound with a width of about 120 μm on the cell layer, and the medium was carefully removed. After washing with DMEM containing 1% FCS, 250 μL of the above-mentioned medium, crude purified Gn. Sample B diluted 500 times with Gn-bi-GP or the above medium was added. After culturing for 24 hours, it was fixed with PBS (-) containing 4.0% by volume paraformaldehyde, and observed with a phase contrast microscope.

  As a result, when only the medium was added, wound closure was enhanced (cell layer wounds were almost filled with cells), whereas crude purified Gn.Gn-bi- Wound closure was suppressed in the GP addition group, and was further suppressed in the sample B addition group containing Gn.Gn.Gn-tri′-glycopeptide.

Enhancement of neurite outgrowth by introduction of GnT-III gene Rat GnT-III cDNA [Nishikawa, A. et al., Journal of Biological Chemistry, Vol. 267, pp. 18199-18204, 1992] .1 (manufactured by Invitrogen). The resulting plasmid was introduced into Neuro-2A cells using Lipofectamine 2000 (Invitrogen). Thereafter, transformed cells were selected using G418 resistance as an index, and a plurality of cell lines stably overexpressing GnT-III were established.

  As a control, a cell line (called Mock) into which a plasmid plasmid pcDNA3.1 not containing GnT-III cDNA was introduced was established in the same manner.

In order to obtain a low density, the same number of GnT-III gene-introduced cell lines and Mock cell lines were seeded in 35 mm glass bottom culture dishes, respectively, and high-glucose type DMEM medium (manufactured by SIGMA) containing 10 wt% FCS. Culturing was carried out for 12 hours at 5% by volume CO ₂ .

  Then, differentiation induction by serum starvation was performed by replacing the medium with a DMEM medium not containing FCS. As a control, a group was also tested that continued to culture both cell lines in media containing FCS. After culturing for 24 hours, the cells were fixed with PBS containing 4% by volume of formaldehyde for 10 minutes. The fixed cells were treated with Alexa 546-phalloidin for the actin cytoskeleton, anti-phosphorylated NF-H mouse monoclonal antibody SMI-31 and Alexa 546-labeled goat anti-mouse IgG antibody for NF-H, which is a neuronal differentiation marker. Double staining was performed using a general indirect fluorescent antibody staining method, and images were taken with a confocal laser microscope.

  Count the total number of cells and the number of NF-H positive cells in the laser microscope field of view (73.1 × 73.1 μm), NF-H positive (extended protrusions are stained with anti-NF-H antibody) ) Was evaluated as a percentage of the total number of cells. Three different clones for each of the GnT-III gene-introduced cell line and Mock were used in the experiment, and the average of these clones is shown in FIG. In the figure, + and-indicate the presence or absence of FCS, respectively.

  As a result, as shown in FIG. 2, it became clear that neurite outgrowth from Neuro-2a cells accompanied by differentiation induction by serum starvation is enhanced by overexpression of the GnT-III gene.

  The above results strongly suggest that the GnT-III gene product is deeply involved in the neurite outgrowth process, among neural differentiation.

  The glycopeptide (neurite outgrowth inducer) obtained in Example 2 is mixed with a solution containing sterile physiological saline and an auxiliary agent for injection under sterile conditions to treat the neurological disease in an injection form. Or a preventive agent is obtained.

  The agent for treating or preventing a neurological disease is administered to an individual's nerve injury site. Over time, the motor ability, speech ability, and / or the degree of recovery of response to external stimuli that are impaired due to the nerve damage is evaluated.

  As a result, when the motor ability, speech ability, and / or response to external stimuli that have been impaired due to nerve damage are recovered, it is evaluated as an indicator that a pharmacological effect by a therapeutic or prophylactic agent for neurological diseases has been obtained. .

  The glycopeptide (neurite outgrowth inducer) obtained in Example 2 is mixed together with the raw materials in the production of beverages and foods.

  The obtained beverage or food is supplied to a normal individual and an individual whose learning ability and / or memory ability are reduced due to nerve damage. Then, the learning ability and the memory ability before and after the supply of the beverage or food are tested, and the results are evaluated.

  Thereby, when the result of a normal individual improves after supply of a drink or food compared with before supply of a drink or food, it becomes an index that an improvement effect of learning ability and memory ability is seen by the drink or food. In addition, when the performance of an individual whose learning ability and / or memory ability has decreased due to nerve damage after the supply of the beverage or food is close to the normal level after the supply of the beverage or food, the learning ability and It is an indicator that memory memory can be improved.

  According to the present invention, a sugar chain having a bisecting GlcNAc, a complex carbohydrate, a derivative thereof, a salt thereof, an enzyme related to the synthesis of the sugar chain, and a medicine, a reagent, a food or a beverage containing a nucleic acid encoding the enzyme Is provided. The medicine, food or beverage has a neurite outgrowth-inducing action of a sugar chain having a bisecting GlcNAc, a complex carbohydrate, a derivative thereof and / or a salt thereof, to treat or prevent a neurological disease, or to improve or improve a learning memory ability Useful for. The present invention also provides a reagent having a neurite outgrowth-inducing action containing the active ingredient.

  SEQ ID NO: 1 shows the amino acid sequence of the peptide part of the glycopeptide.

Claims (22)

  A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, and the complex carbohydrate A neurite outgrowth inducer comprising, as an active ingredient, at least one selected from the group consisting of a pharmacologically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. A sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The neurite outgrowth inducer according to claim 1, wherein A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
The neurite outgrowth inducer according to claim 1, wherein   A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, and the complex carbohydrate A therapeutic or preventive agent for neurological diseases comprising, as an active ingredient, at least one selected from the group consisting of a pharmacologically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme Agent. A sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The therapeutic agent or preventive agent for neurological disease of Claim 4 which is a sugar chain represented by these. A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
The therapeutic agent or preventive agent for neurological disease of Claim 4 which is a sugar chain represented by these.   The therapeutic or preventive agent for neurological diseases according to any one of claims 4 to 6, which is used for the treatment or prevention of neurodegenerative diseases, dementia or brain tumors.   A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, and the complex carbohydrate A learning and / or memory ability comprising as an active ingredient at least one selected from the group consisting of a pharmacologically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme Improver or improver. A sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The learning and / or memory ability improver or improver according to claim 8, wherein the sugar chain is represented by: A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
The learning and / or memory ability improver or improver according to claim 8, wherein the sugar chain is represented by:   A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, and the complex carbohydrate A food or beverage comprising at least one selected from the group consisting of pharmacologically acceptable salts. A sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The food or beverage according to claim 11, which is a sugar chain represented by the formula: A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
The food or beverage according to claim 11, which is a sugar chain represented by the formula:   The food or beverage according to any one of claims 11 to 13, which is used for treatment or prevention of neurodegenerative diseases, dementia or brain tumors.   The food or beverage according to any one of claims 11 to 13, which is used for improving or preventing learning and / or memory ability.   A sugar chain having bisecting GlcNAc, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, and the complex carbohydrate Neurite outgrowth characterized by administering to a specimen at least one selected from the group consisting of a pharmacologically acceptable salt, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme Guidance method. The sugar chain having bisecting GlcNAc is represented by the following formula (1):

(In the formula, R ¹ represents a sugar residue, and ± means the presence or absence of a GlcNAc residue)
The method for inducing neurite outgrowth according to claim 16, wherein A sugar chain having bisecting GlcNAc is represented by the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
The method for inducing neurite outgrowth according to claim 16, wherein To an individual suffering from a neurological disease or an individual who is likely to suffer from the neurological disease, the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
A sugar chain having a bisecting GlcNAc represented by: a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, A neurological disease characterized by administering at least one selected from the group consisting of a pharmacologically acceptable salt of the glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Treatment or prevention method. For individuals who need to improve or improve their learning ability, the following formula (2):

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. Indicates
A sugar chain having a bisecting GlcNAc represented by: a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, Supplying at least one selected from the group consisting of a pharmacologically acceptable salt of the glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme, and // Method for improving or improving memory capacity. The following formula (2) for the manufacture of a medicament for the treatment or prevention of neurological diseases:

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
A sugar chain having a bisecting GlcNAc represented by: a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, Use of at least one selected from the group consisting of a pharmacologically acceptable salt of the glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Formula (2) for the production of an enhancer or improver of learning and / or memory ability:

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are GlcNAc residues optionally bonded to mannose via a β1-2 bond, a β1-4 bond, or a β1-6 bond. (However, when R ² and R ³ have a GlcNAc residue, 1 or 2 R ² and R ³ may be directly bonded to mannose)]
A sugar chain having a bisecting GlcNAc represented by: a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex carbohydrate, a pharmacologically acceptable salt of the sugar chain, Use of at least one selected from the group consisting of a pharmacologically acceptable salt of the glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme.

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