WO2005097155A1

WO2005097155A1 - Neurite elongation inducing agent

Info

Publication number: WO2005097155A1
Application number: PCT/JP2005/006640
Authority: WO
Inventors: Jianguo Gu; Masaki Shigeta; Naoyuki Taniguchi
Original assignee: Takara Bio Inc.
Priority date: 2004-04-08
Filing date: 2005-04-05
Publication date: 2005-10-20
Also published as: JPWO2005097155A1

Abstract

Exhibiting a high neurite elongation effect, a novel neurite elongation agent, therapeutic or preventive agent for neurodegenerative disorder, food or drink for the treatment or prevention of this disorder, method of inducing neurite elongation, method of treating or preventing neurological disorder, method of enhancing or improving learning and/or memorizing capacity, use of sugar chain in the production of medicine for the treatment or prevention of neurological disorder, and use of sugar chain in the production of learning and/or memorizing capacity enhancer or improver.

Description

Specification

Neurite outgrowth inducer

Technical field

The present invention relates to a sugar chain having a neurite elongation effect, a complex saccharide, a derivative of the sugar chain or the complex saccharide, a pharmacologically acceptable salt of the sugar chain or the complex saccharide, or a pharmaceutically acceptable salt thereof. The present invention relates to an enzyme involved in sugar chain synthesis and a pharmaceutical composition, reagent, food or beverage containing a nucleic acid encoding the enzyme as an active ingredient.

Background art

[0002] Nerve cells usually construct a complex network by extending one axon and a plurality of 榭 processes and exchanging signals with other cells. A series of substances called neurotrophic factors play an important role in maintaining neuronal cell functions such as survival of neurons, differentiation of immature neuroblasts into mature neurons, neurite outgrowth, etc. Is believed to be.

[0003] Diseases associated with nerve injury or neurodegeneration, such as nerve transection, spinal cord injury, diabetic neuropathy, amyotrophic lateralsclerosis (ALS), and multiple sclerosis (MS) Treatment of peripheral nervous system and central nervous system diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease includes methods to promote the survival and repair of damaged nerve cells (preservation regeneration) and neuronal cells. It is roughly classified into a method of replenishing cells capable of differentiating into cells (for example, neural stem cells) at the site of injury and recovering function (replenishment regeneration). Among them, the above-mentioned replenishment regeneration has not yet become widespread due to difficulties in obtaining cells to be replenished and ethical problems. On the other hand, the preservation regeneration is, more specifically, a method of restoring function by regenerating axons from remaining nerve cells and forming new synapses.

[0004] In recent years, attention has been focused on neurotrophic factors for the purpose of preventing and treating neurodegenerative diseases. For example, nerve growth factor (nerve growth factor, NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3, Neurotrophin-4Z5 and the like have been found. These nerve growth factors It is promising as a therapeutic drug for neurological dysfunction due to neuronal damage or as a preventive drug for dementia (see, for example, Non-Patent Document 1).

[0005] For example, Patent Document 1 reports a neurotrophic factor having a molecular weight of 60,000 5,000 and a neurotrophic factor having a molecular weight of 120,000 5,000 obtained from a culture medium for astrocytoma, glial cells or Schwann cells. RU The neurotrophic factor described in the powerful Patent Document 1 has a neuroblastoma growth inhibitory activity and has a survival activity or a neurite outgrowth effect on nerve cells. Furthermore, Patent Document 2 reports a glycosaminodalican derivative having a neurite outgrowth action with low anticoagulant activity.

[0006] However, since these proteinaceous neurotrophic factors and polysaccharide derivatives are high-molecular-weight compounds, they can reach trophic factors to affected areas in the brain beyond barriers such as the blood-brain barrier. It is extremely difficult, and there is a problem that the effect of the proteinaceous neurotrophic factor, polysaccharide derivative and the like is greatly affected by the administration method.

[0007] On the other hand, low-molecular-weight neurotrophic factors include, for example, a neurite elongation agent containing a polyalkoxyflavonoid extracted from Rutaceae plants (Patent Document 3), a nerve cell proliferation action and a neurite extension action. For example, a cyclohexenol derivative (Patent Document 4) and a neurite outgrowth agent containing a pyrrolidine derivative or a piperidine derivative exhibiting a nerve cell growth promoting action (Patent Document 5) have been reported. However, at present, these substances are not sufficiently effective.

Patent document 1: JP-A-07-101990

Patent Document 2: JP-A-11-310602

Patent Document 3: JP 2002-060340A

Patent Document 4: JP 2001-089404 A

Patent Document 5: Japanese Patent Laid-Open No. 2001-247569

Non-Patent Document 1: "Nature", Vol. 344, 339-341 (1990) Disclosure of the Invention

Problems to be solved by the invention

[0008] The present invention relates to a novel neurite outgrowth agent, a therapeutic or preventive agent for neurodegenerative disease, a food or drink for treating or preventing the disease, and a nervous elongation effect which exerts a neurite outgrowth effect. A method for inducing process elongation, a method for treating or preventing a neurological disease, a method for improving or improving learning and Z or memory ability, a method for using a sugar chain for the manufacture of a medicament for treating or preventing a neurological disease, and a method for learning and preventing. Z or a use of the sugar chain for the manufacture of a memory capacity improver or improver. In one aspect, the present invention relates to providing a neurite outgrowth inducing agent that enables a new neural network to be formed based on neurite outgrowth. In another aspect, the present invention relates to providing a therapeutic or preventive agent for a neurological disease, which is capable of ameliorating the symptoms of a disease associated with neurodegeneration, suppressing the onset or progression of the disease, and the like. . In still other aspects, the present invention provides a learning, which allows for the enhancement of the learning and Z or memory capacity of an individual animal, the learning and recovery of the Z or memory capacity of an individual that has been reduced by various factors, and the like. And Z or providing a memory improver or improver. In another aspect, the present invention relates to the provision of a food or beverage which, when ingested, can provide a therapeutic or preventive effect for a neurological disease, and an effect of improving or improving learning and Z or memory ability. In yet another aspect, the present invention relates to providing a method of inducing neurite outgrowth, which allows for the generation of neurites that differentiate into axons or neurites and to induce Z or elongation, and the like. In another aspect, the present invention provides a method for treating or preventing a neurological disease, which is capable of improving symptoms of a disease accompanied by neurodegeneration, suppressing the onset or progression of the disease, and the like. Use of a prescribed sugar chain for the manufacture of a medicament for the treatment or prevention of a neurological disease, which enables the supply of a medicament suitable for performing a method for treating or preventing a neurological disease, and the treatment or prevention of a neurological disease. Related to providing. In another aspect,

The present invention provides a learning and Z or memory function that enables the learning and Z or memory capacity of an individual animal to be extended, the learning and the z or memory capacity of the individual to be reduced due to various factors, and the like. A method for improving or improving the ability, and supplying a learning and Z or memory improving agent or improving agent suitable for performing the method for improving or improving the learning and Z or memory ability, and improving the learning and Z or memory ability in the individual. Or the use of defined sugar chains for the production of learning or Z or memory abilities improvers, or the like. Note that the other objects of the present invention are derived from the description in this specification. Means for solving the problem

That is, the first invention of the present invention relates to a sugar chain having bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, the sugar chain or a derivative of the complex sugar, the sugar chain or Comprises, as an active ingredient, 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. Related to inducers.

[0010] The second invention of the present invention relates to a sugar chain having bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain or the complex saccharide, the sugar chain or the complex saccharide. Remedy or prevention for a neurological disease, comprising as an active ingredient at least one selected from the group consisting of a pharmacologically acceptable salt of high quality, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Agent. Examples of the neurological disease include neurodegenerative diseases, dementia, and brain tumors.

[0011] The third invention of the present invention relates to a sugar chain having bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain or the complex saccharide, the sugar chain or the complex saccharide. Quality and pharmacologically acceptable salts, an enzyme involved in the synthesis of the sugar chain, and at least one selected from the group consisting of nucleic acids encoding the enzyme as an active ingredient, having learning and Z or memory ability. It relates to an enhancer or an improver.

[0012] A fourth invention of the present invention provides a sugar chain having bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain or the complex saccharide, the sugar chain or the complex saccharide. The present invention relates to a food or beverage containing at least one member selected from the group consisting of a pharmacologically acceptable salt of high quality, an enzyme involved in synthesizing 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 a neurodegenerative disease, dementia or brain tumor, or the treatment or prevention of the neurodegenerative disease, dementia or brain tumor. Food or beverages to assist in prevention, food or beverages used to improve or improve learning and Z or memory ability, foods or beverages to assist in improving or improving learning and Z or memory ability, etc. Is exemplified.

[0013] A fifth invention of the present invention relates to a sugar chain having bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain or the complex saccharide, the sugar chain or the complex saccharide. 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. The method of inducing.

[0014] In the first to fifth inventions of the present invention, the sugar chain having bisecting GlcNAc

, The following equation (1):

[0015] [Chemical 1] GlcNAcpl-2Manal \

0

GlcNAc l-4Man lR ¹ (1)

Three

Sat GlcNAcpi-2Mantxl '

(Wherein, R ¹ represents a sugar residue, and “士” means the presence or absence of a GlcNAc residue)

Are exemplified.

That is, the gist of the present invention is as follows:

(1) bisecting a sugar chain having GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex saccharide, a pharmacologically acceptable salt of the sugar chain, A neurite comprising as an active ingredient at least one selected from the group consisting of a pharmacologically acceptable salt of a glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Elongation inducer,

[2] Bisecting GlcNAc-containing sugar chain force Formula (1) below:

[0018] [Chemical 2] Sat GlcNAcpl-2Manak

GlcNAcpi-4Manpl-Ri (1)

Three

Sat GlcNAceijManal ⁷

[0019] (In the formula, R ¹ represents a sugar residue, and R means the presence or absence of a GlcNAc residue.)

Is a sugar chain represented by the neurite outgrowth inducing agent according to (1),

[3] Bisecting GlcNAc-containing sugar chain force Formula (2) below: [0020] [Formula 3]

R ² -Manal \

6

GlcNAcpi-4Man lR ¹ (2)

Three

R ³ -Manod,

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ bind to mannose via a β 1-2 bond, a β 1-4 bond or a 1-6 bond. Indicates a GlcNAc residue (however, when R ² and R 3 have a GlcNAc residue, one or two R ² and R ³ may be directly bonded to mannose)]

(4) a bisecting sugar chain having GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, a pharmacologically acceptable salt of the sugar chain, A neurological disease comprising, as an active ingredient, at least one selected from the group consisting of a pharmacologically acceptable salt of a glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Therapeutic or prophylactic agents,

[5] Bisecting Sugar chain strength with GlcNAc Formula (1)

[0022] [Chemical 4] Soil Soil

In the sugar chain represented by, the therapeutic agent or preventive agent for a neurological disease according to (4),

[6] Bisecting Sugar chain force with GlcNAc Formula (2) [0024] [Formula 5]

R ² -Manal \

6

GlcNAcpl ^ Manpl-R ¹ (2)

Three

R ³ -Manotl,

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are linked to mannose via a β 1-2 bond, a β 1-4 bond or a 1-6 bond. Indicates a GlcNAc residue (however, when R ² and R 3 have a GlcNAc residue, one or two R ² and R ³ may be directly bonded to mannose)]

[7] used for the treatment or prevention of neurodegenerative disease, dementia or brain tumor, the above-mentioned [4]

(6) the therapeutic or prophylactic agent for a neurological disease according to any one of the above,

(8) bisecting a sugar chain having GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex saccharide, a pharmacologically acceptable salt of the sugar chain, Learning and learning comprising at least one selected from the group consisting of a pharmacologically acceptable salt of a glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme as an active ingredient. Z or memory improver or improver,

[9] Bisecting Sugar chain strength with GlcNAc Formula (1)

[0026] [Formula 6]

Sat GlcNAcpl-2M

(GlcNAcpl-AManpl-R ¹ fl)

Three

Sat GlcNAcei-SMana ⁷

A learning or Z or memory improving or improving agent according to the above (8), which is a sugar chain represented by

[10] Bisecting Sugar chain force with GlcNAc Formula (2) [0028]

R ² -Mana

6

GlcNAcpi ^ Manpi-R ¹ (2)

Three

R ^ Manal

[In the formula, R ¹ represents a sugar residue, and R ² and R ³ are bonded to mannose via a β 1-2 bond, a β 1-4 bond or a 1-6 bond. Indicates a GlcNAc residue (however, when R ² and R 3 have a GlcNAc residue, one or two R ² and R ³ may be directly bonded to mannose)]

(11) bisecting a sugar chain having GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex saccharide, a pharmacologically acceptable salt of the sugar chain, and A food or beverage comprising at least one member selected from the group consisting of pharmacologically acceptable salts of glycoconjugates;

[12] Bisecting Sugar chain strength with GlcNAc The following formula (1):

[0030] [Formula 8]

± GlcNAc l-2Man l \

6

GlcNAc i ^ Man iR ¹ (1)

Three

GlcNAcpUMancdZ

(In the formula, R ¹ represents a sugar residue, and “1” means the presence or absence of a GlcNAc residue.)

In the sugar chain represented by the food or beverage according to the (11),

[13] Bisecting Sugar chain strength with GlcNAc The following formula (2):

[0032]

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ represent a β 1-2 bond, a β 1-4 bond or 1-6 Indicates a GlcNAc residue which may be bound to mannose via a bond (however, when R ² and R 3 have a GlcNAc residue, one or two R ² and R ³ are directly bound to mannose; May be)

In the sugar chain represented by the food or beverage according to the (11),

(14) a neurodegenerative disease, used for treatment or prevention of dementia or brain tumor, the (11) food or beverage according to any one of (13),

[15] The food or beverage according to any one of the above [11] to [13], which is used for improving or preventing learning and Z or memory ability.

(16) a sugar chain having bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex saccharide, a pharmacologically acceptable salt of the sugar chain, Administering at least one selected from the group consisting of a pharmacologically acceptable salt of a glycoconjugate, an enzyme involved in synthesizing the sugar chain, and a nucleic acid encoding the enzyme. Neurite outgrowth induction method,

[17] Sugar chain having bisecting GlcNAc The sugar chain having bisecting GlcNAc is represented by the following formula (1):

[0034] [Chemical Formula 10] Sat GlcNAcp l-2M 1 \

o

GlcNAcpl-4Manpl-R ¹ (1)

Three

GlcNAcpl-SManal ⁷

A sugar chain represented by the method of inducing neurite outgrowth according to the above (16),

[18] Bisecting Sugar chain with GlcNAc Formula (2)

[0036] [Formula 11]

GlcNAcpl-4Man iR ¹ (2)

Three

R ³ -Manal '

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ represent a β 1-2 bond, a β 1-4 bond or 1-6 bond. Indicates a GlcNAc residue which may be bound to mannose via a bond (however, when R ² and R 3 have a GlcNAc residue, one or two R ² and R ³ are directly bound to mannose; May be)

[19] The following formula (2) is given to an individual suffering from a nerve disease or an individual at risk of suffering from the nerve disease.

[0038] [Formula 12]

R ² -Manalv

6

GlcNAc l- AManpl-R ¹ (2)

Three

R ³ -Manal

A sugar chain having a bisecting GlcNAc represented by: a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, a pharmacologically acceptable salt of the sugar chain, 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. Disease treatment or prevention methods,

[20] For an individual in need of improvement or improvement in learning ability, the following formula (2):

[0040] [Formula 13]

R ² -Man l \

6

GlcNAcpi- AManpi-R ¹ (2)

Three

R ³ -Manal ⁷

[Wherein, R ¹ represents a sugar residue, R ² and R ³ bind to mannose via a β 1-2 bond, a β 1-4 bond or a 1-6 bond, May indicate a GlcNAc residue)

A sugar chain having bisecting GlcNAc represented by the formula, a complex sugar 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 the complex carbohydrate, an enzyme involved in the synthesis of the sugar chain And a method for improving or improving learning and Z or memory ability, characterized by supplying at least one selected from the group consisting of nucleic acids encoding the enzyme.

[21] The following formula (2) for the manufacture of a medicament for treating or preventing a neurological disease:

[0042] [Formula 14]

R ² -Man l \

6

GlcNAc i ^ Manpi-R ¹ (2)

Three

R ³ -ManodZ

A sugar chain having a bisecting GlcNAc represented by: a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, 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 synthesizing the sugar chain, and a nucleic acid encoding the enzyme; and

[22] The following formula (2) for the production of an enhancer or an enhancer for learning and Z or memory ability.

[0044] [Formula 15]

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are bonded to mannose via a β 1-2 bond, a β 1-4 bond or a 1-6 bond. Indicates a GlcNAc residue (however, when R ² and R 3 have a GlcNAc residue, one or two R ² and R ³ may be directly bonded to mannose)] A sugar chain having a bisecting GlcNAc represented by: a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, a pharmacologically acceptable salt of the sugar chain, Use of at least one member 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 invention's effect

According to the present invention, there is provided a medicine, reagent, food or beverage containing a sugar chain having bisecting GlcNAc, a glycoconjugate, a derivative thereof, and Z or a salt thereof. In other words, the neurite outgrowth inducing agent of the present invention has an excellent effect that a new neural network can be formed based on neurite outgrowth. Further, according to the therapeutic or preventive agent for a neurological disease of the present invention, it is possible to ameliorate the symptoms of a disease accompanied by neurodegeneration, and to suppress the onset or progression of the disease. I do. Furthermore, according to the learning and Z or memory abilities improver or improving agent of the present invention, the learning and Z or memory abilities of an individual animal can be extended, and the learning and Z of a reduced individual due to various factors can be improved. It has an excellent effect that Z or memory ability can be recovered. ADVANTAGE OF THE INVENTION According to the food or beverage of the present invention, an excellent effect is obtained in which ingestion of the food or beverage provides a therapeutic or preventive effect for a neurological disease, and an improvement or improvement effect of learning and Z or memory ability. Further, according to the method for inducing neurite outgrowth of the present invention, there is an excellent effect that it is possible to induce the generation of neurites that differentiate into axons or 榭 processes and to induce Z or elongation. ADVANTAGE OF THE INVENTION According to the method for treating or preventing a neurological disease of the present invention, it is possible to improve the symptom of a disease accompanied by neurodegeneration and to suppress the onset or progress of the disease. Furthermore, according to the method for improving or improving the learning and Z or memory ability of the present invention, the learning and Z or memory ability of an animal individual can be extended, and the learning and z of an individual who has decreased due to various factors can be improved. Or, it has an excellent effect that the memory ability can be restored. In addition, according to the use of the specified sugar chain for the manufacture of a medicament for treating or preventing a neurological disease of the present invention, the supply of a medicament suitable for performing the above-mentioned method for treating or preventing a nervous disease, the use of a nervous system, It has an excellent effect of enabling treatment or prevention of a disease. In addition, the learning and Z or According to the use of defined sugar chains for the production of enhancers or improvers, said learning and

Good learning and Z or memory improving agent or supply of improver or improving agent suitable for performing the method of improving or improving Z or memory ability, excellent effect of enabling learning and improving or improving Z or memory ability in an individual. To play.

Brief Description of Drawings

FIG. 1 is a diagram showing the neurite outgrowth-inducing effect of Gn (Gn) Gn-W-GP. In the figure

, Open bars indicate nerve extension and black bars indicate cell extension.

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

BEST MODE FOR CARRYING OUT THE INVENTION

[0049] The present invention provides a sugar chain having a bisecting GlcNAc (bisecting GlcNAc) structure, a glycoconjugate having the sugar chain structure, and an enzyme relating to the synthesis of the sugar chain, which exhibits an excellent neurite elongation effect. Based on their surprising findings.

[0050] In one aspect, the present invention relates to a neurite outgrowth inducing agent.

[0051] The neurite outgrowth inducing agent of the present invention includes a sugar chain having the bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, the sugar chain or a derivative of the complex sugar, the sugar chain or the sugar chain. One major feature is that it contains as an active ingredient at least one selected from the group consisting of a pharmacologically acceptable salt of a glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. And Therefore, according to the neurite outgrowth-inducing agent of the present invention, a high neurite outgrowth-inducing effect can be obtained in a site where neurite outgrowth is desired, for example, a site of nerve damage or a site where neurodegeneration is observed. In addition, it has an excellent effect that neurites can be efficiently elongated.

[0052] In the differentiation of undifferentiated neurons, neurites elongate first, and then one of the neurites rapidly elongates to exhibit axonal properties, In addition, the remaining neurites acquire the properties of 榭. The axons and dendrites of the cells thus formed form synapses with other cells to form a neural circuit. According to the neurite outgrowth inducer of the present invention, the induced neurite force axons or 突起 processes are generated, and an excellent effect of promoting the formation of a new neural network is exhibited. Further, according to the neurite outgrowth inducing agent of the present invention, the neurite is elongated by extending the neurite. It is possible to regenerate vesicles, treat and prevent neurological diseases such as neurodegenerative diseases, and the like.

[0053] In general, a sugar chain of a glycoprotein is bound to an asparagine residue of the protein to form an N-glycoside-linked sugar chain (also referred to herein as "N-darican") and a serine chain of the protein. O-glycoside-linked sugar chains (also referred to as “0-darican”) linked via the hydroxyl group of the residue or threonine residue. The N-darican has a reducing terminal sugar residue, N-acetyldarcosamine (GlcNAc), bonded to an amino group of asparagine on the peptide chain, and a reducing terminal N-acetyldarcosamine has an anomeric hydroxyl group. To form a bond state of [sugar chain-NH-CO-CH-CH (NH) COOH]

twenty two

[0054] N-glycans are further referred to as complex-type sugar chains comprising sugar residues such as sialic acid, galactose, mannose, fucose, and N-acetyl-darcosamine ("N-acetyl-lactosamine-type sugar chains"). An oligomannose-type sugar chain comprising mannose (Man) and N-acetyl-darcosamine, and a mixed molding having a structure in which the N-acetyl-lactosamine-type sugar chain and the oligomannose-type sugar chain are combined. Sugar chains (also referred to as "hybrid-type sugar chains") are classified into three types.

[0055] The sugar chain having bisecting GlcNAc in the present invention is a sugar chain classified as the above-mentioned complex type sugar chain or hybrid sugar chain, and the hydroxyl group at the 4-position of the β-mannose residue of the sugar chain. And a sugar chain having acetylacetylcosamine bonded to glycerol.

The sugar chain having a complex type bisecting GlcNAc that can be used in the present invention includes, for example, the following formula (1):

[0057] [Formula 16] Sat GlcNAcpl-2Manal \

6

GlcNAcpl-AManpl-R ¹ (1)

Three

Sat GlcNAcpl-2Manal '

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

And the like.

[0059] The sugar chain of the formula (1) is, in other words, the following formula (2): [0060] [Formula 17]

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ bind to mannose (Man) via a β 1-2 bond, a β 1-4 bond or a 1-6 bond. Represents a GlcNAc residue (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 (Man)) ]

Is a sugar chain included in the sugar chain represented by

[0062] Specifically, for example, the following formula (3):

[0063]

GlcNAcpl-4Man iR ³ f3)

Three

GlcNAc i-2ManaT R ³

[Wherein, R ¹ represents an arbitrary sugar residue, and R ² represents a GlcNAc residue which may be bound to mannose (Man) via a β ^1-6 bond or a β 1-4 bond. R ³ represents a GlcNAc residue which may be bound to mannose (Man) via a j81-4 bond or | 81-6 bond].

[0065] More specifically, as the sugar chain, for example, the following formula (4):

[0066] [Formula 19]

GlcNAcpl-2Man l \

6

GlcNAc i-4Man lR ¹ (4)

Three

GlcNAc i-2Man l

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

[0068] [Formula 20]

GlcNAcp l-2M 1 \

6

GlcNAc i-4Manpi-R ¹ (5)

Three

GlcNAceUManctl

Four

GlcNAcpi,

(Wherein, R ¹ represents a sugar residue)

Or

[0070]

GlcNAcP K

6

GlcNAcpi-2Manal

GlcNAc i-4Man iR ¹ (6)

Three

GlcNAcpUManctlZ

(Wherein, R ¹ represents a sugar residue)

A sugar chain having a three-chain structure represented by the following formula (7)

[0072]

GlcNAc K

GlcNAcpl-4Man iR ¹ (Ί)

Three

GlcNAc l-ZManal ⁷

Four

GlcNAcpl,

(Wherein, R ¹ represents a sugar residue)

A sugar chain having a four-chain structure represented by is exemplified. In the present invention, when synthesizing a sugar chain enzymatically, a sugar chain having a double-chain structure can be preferably used from the viewpoint of ease of production.

[0074] The sugar chains used in the present invention include, in addition to the complex type sugar chains described above, bisecting GlcN Mixed sugar chains having Ac are also exemplified. Examples of the hybrid sugar chains having Neusecting GlcNAc used in the present invention include sugar chains having a double-chain structure, sugar chains having a triple-chain structure, sugar chains having a 4-chain structure, and the like.

[0075] As the sugar residue represented as R ¹ in the sugar chain represented by the formula (1) to (7), for example, G1 CNAC residue, glucose residue, fucose residue, a sialic acid residue Group, N-acetylgalatatosamine residue, galactose residue, mannose residue, N-acetylmannosamine residue, xylose residue and the like.

[0076] R ² and R ³ in the sugar chain represented by the formula (2) shows the GlcNAc residues. R ² and R ³ may be bonded to a mannose residue via a j8 1-2 bond, | 8 1-4 bond or | 8 1-6 bond according to the above formula (2). It may not be present in the sugar chain to be prepared. 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. Two R ² are, when attached to one mannose residue, wherein the two R ² may bind to a mannose residue at different binding modes. On the other hand, when R ³ is present in the sugar chain represented by the formula (2), the R ³ also has one or two Rs per one mannose residue similarly to the R ^2. ³ forces can be combined. Also, two R ^3, when attached to one mannose residues, as well as the R ^2, the two R ³ binds to mannose residues in different binding modes.

[0077] The sugar chain having Neusecting GlcNAc used in the present invention is represented by the above formula

(4) to (6) The ability to give a sugar chain having a non-reducing end of N-acetyltylcorsamine as long as it exhibits the desired effect in the present invention. Not something. That is, the sugar chain having bisecting GlcNAc used in the present invention is a sugar chain in which the non-reducing terminal side of the sugar chain is further modified with galactose, sialic acid, fucose, glucuronic acid, GlcNAc, mannose, or the like. Is also good.

[0078] In the present invention, the glycoconjugate having a bisecting GlcNAc in its structure is a glycoconjugate having a bisecting GlcNAc added to any protein, peptide, lipid, or the like. Are shown. That is, glycoconjugates having a sugar chain having Neusecting GlcNAc in the structure used in the present invention include glycopeptides, glycoproteins, glycolipids and the like. [0079] 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. Is not particularly limited as long as a sugar chain or complex carbohydrate exhibiting the desired effect can be obtained.For example, natural sugars or complex carbohydrates having no bisecting GlcNAc obtained naturally are artificially produced. Secting A method of introducing GlcNAc is exemplified. The introduction of bisecting GlcNAc is not particularly limited, but can be performed enzymatically using N-acetyl darcosamyltransferase ΠΙ (also referred to herein as “GnT-III”).

[0080] Hereinafter, the introduction of enzymatic bisecting GlcNAc by GnT-III will be described in detail. First, a sugar chain or complex carbohydrate serving as a substrate for GnT-III is obtained.

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

When a glycopeptide is obtained as the glycoconjugate used as the substrate, the glycoprotein described above is subjected to limited protein hydrolysis using an enzyme such as a protease such as trypsin, lysyl endopeptidase, or chymotrypsin. To obtain a glycopeptide. When a glycoprotein is degraded by a protein-limited hydrolysis with a protease, an enzymatic reaction is performed in a dialysis membrane or an ultrafiltration membrane, whereby the glycopeptide produced by the degradation with the enzyme can be easily separated. Purification of the target glycopeptide from the mixture of degraded peptides, degraded peptides and glycopeptides by purification methods such as centrifugation, gel filtration, ion exchange chromatography, hydrophobic chromatography, and lectin affinity chromatography can do.

[0083] As the substrate, free glycopeptides present in eggs of mammals such as eggs, birds and fish can also be used. 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, and lectin affinity chromatography.

. An example of obtaining a glycopeptide from an egg will be described below. -After adding the same amount of water to the chicken egg yolk, add 1 to 10 phenol-water (9: 1) mixture and stir well. Incidentally Then, the obtained mixture is subjected to centrifugation to collect the supernatant. After concentrating the obtained supernatant, gel filtration chromatography using Sephadex G-50 is carried out to obtain a fraction which is positive by the neutral sugar color reaction by the phenol sulfate method. As a result, a glycopeptide having a double-stranded complex type sugar chain can be obtained. The structure of the glycopeptide (SEQ ID NO: 1) thus obtained is shown in the following formula (8).

[0084]

Lys (NH ₂ )

Neu Ac 2-6Gaip 1 -4GlcNAcP 1 -2Manal \ Val

6 Ala

Manpi-4GlcNAcpi-4 GlcNAc i-Asn

3 Lys

Ν _εϋ Α _ε α2-6θ3ΐ 1-401οΝΑςβ1-2Μ ₃ ηα1 Thr (COOH)

(8)

[0085] When a sugar chain to be used as a substrate is obtained, the glycoprotein power of the calf serum _futein , _transferrin , _Ί -globulin and the like is also measured by a chemical method such as hydrazinolysis {glycopeptidase} (almond). Complex sugars using an enzymatic technique using, for example, origin, for example, Seikagaku Corporation), glycopeptidase F (derived from flavata terium, also referred to as "PNGase", for example, Takara Bio Inc.). Can release chains (Glycoprotein sugar chain research method, Biological Chemistry Experimental Method 23, published by Gakkai Shuppan Center, 1989).

[0086] When GnT-III is allowed to act on the sugar chain or glycoconjugate obtained as described above, usually, sialic acid, galactose residue or fucose residue is removed from these non-reducing ends. I do. A known method may be used to remove the sugar residue at the non-reducing end.For example, sialic acid may be removed in a dilute solution of an inorganic acid such as dilute hydrochloric acid or dilute sulfuric acid at 50 ° C. Sialic acid is liberated by heating at 100 ° C for 10 minutes to 2 hours, preferably in a 0.025-0.1N hydrochloric acid solution or a 0.025-0.1N sulfuric acid solution at 80 ° C for 1 hour. This can be done. When removing a sugar residue by an enzymatic method, a sialic acid residue, a galatatose residue, a fucose residue and the like are removed by an enzymatic treatment with sialidase, galactosidase, fucosidase or the like. This makes it possible to obtain a sugar chain or complex saccharide whose non-reducing end is GlcNAc, which is a suitable substrate for GnT-III.

[0087] Examples of the sialidase used here include sialidase derived from Arthrobacter ureafaciens and Clostridium nofringe. Enzymes having a wide substrate specificity such as sialidase derived from Clostridium perfringens are preferably used. As the galactosidase, for example, an enzyme having a wide substrate specificity such as j8-galactosidase derived from peanut beans and j8-galactosidase derived from Aspergillus niger is preferably used. As the fucosidase, for example, a-fucosidase derived from Streptomyces sp. 142 (Streptomyces sp. 142) strain, α-fucosidase derived from sea urchin kidney and the like can be used.

[0088] Bisecting GlcNAc is enzymatically introduced by allowing GnT-III to act together with UDP-GlcNAc on the thus obtained sugar chain or glycoconjugate having GlcNAc at the non-reducing end. At this time, if desired, N-acetyl darcosaminyltransferase IV (also referred to as “GnT-IV” in the present specification), N-acetyl darcosamyl-transferase V (in the present specification, “GnT-V ) With UDP-GlcNAc to obtain various sugar chains or complex carbohydrates having a three- or four-chain structure.

As the GnT-III, GnT-IV and GnT-V, the purified enzymes described in Patent No. 2789283, JP-A-6-197756, WO 98Z26053 and the like can be used.

[0090] Further, as the above-mentioned GnT-III, GnT-IV and GnT-V, nucleic acids encoding GnT-III

(Gene), nucleic acid encoding GnT-IV (gene), nucleic acid encoding GnT-V (gene) Use cell lysate, crude enzyme solution or purified enzyme prepared from animal cells etc. transformed with nucleic acid (gene), respectively. You can also. For example, an expression vector for an animal cell having a neomycin resistance gene containing a construct in which a GnT-ΠΙ gene, a GnT-IV gene, or a GnT-V gene is linked downstream of the actin promoter is used for B16 melanoma cells and rat. Transfected cells such as PC12 cells derived from adrenal pheochromocytoma by the electoporation method or lipofectamine, etc., and the resulting cells are screened for GnT- by screening for transformed cells using G418 resistance as an index. Cells expressing III, GnT-IV or GnT-V are obtained. The obtained cells are subjected to sonication, and the supernatant obtained by removing insolubles by centrifugation can be used as a crude enzyme solution. The sugar chain compound used in the present invention and its derivative were synthesized by an enzyme reaction using the crude enzyme solution or the purified enzyme. Thereafter, for example, gel filtration chromatography using trade name: SEPHADEX G15 (manufactured by Amersham's Bioscience), trade name: separation using cellulose cartridge (manufactured by Takara Bio Inc.), from the obtained enzyme reaction solution. Thus, a sugar chain compound or a derivative thereof can be separated and purified from enzymes, sugar nucleotides, salts and the like.

[0091] In the present invention, examples of the sugar chain or complex saccharide having Neusecting GlcNAc include a sugar chain or complex saccharide having a non-reducing terminal force SGlcNAc. In the present invention, the sugar chain or complex saccharide having bisecting GlcNAc is a sugar chain or complex saccharide whose non-reducing terminal is appropriately modified with fucose, galactose, sialic acid, glucuronic acid, GlcNAc, mannose, or the like. It may be quality.

[0092] In addition to the above-mentioned enzyme reactants, sugar chains or conjugates containing bisecting GlcNAc obtained from natural products are included in the sugar chains and complex saccharides having Neusecting GlcNAc used in the present invention. Carbohydrates are also included. As a method for selectively obtaining a sugar chain or complex carbohydrate having Neusecting GlcNAc, it is preferable to use a lectin that specifically binds to a sugar chain having bisecting GlcNAc, such as kidney bean lectin (E4PHA). The method is exemplified. Further, in the present invention, a glycoprotein having bisecting GlcNAc obtained by a known method, for example, γ-daltamyl transpeptidase, γ globulin, etc., is subjected to appropriate treatment so that it can be used as it is. May be used as the glycopeptide or sugar chain. In the sugar chain or glycoconjugate obtained in this manner, the sugar residue on the non-reducing terminal side is not particularly limited. For example, a GlcNAc residue, a galactose residue, a sialic acid residue, a fucose residue, Any sugar residue such as a mannose residue may be used.

[0093] The sugar chain or complex carbohydrate having Neusecting GlcNAc used in the present invention may be a sugar chain or complex carbohydrate produced by synthesis.

[0094] In the present invention, a sugar chain or complex carbohydrate derivative having the above-mentioned bisecting GlcNAc can be used as long as it exhibits a neurite elongation effect. In the present invention, examples of the derivative include conjugates obtained by modifying the sugar chain or complex saccharide within a range in which the desired activity, that is, the neurite elongation effect is not lost. . The modification includes, but is not particularly limited to, the introduction of a non-natural type sugar residue, Examples thereof include addition of a ligated compound or ligands (such as biotin and histidine-tag) and immobilization to a carrier.

[0095] 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, in order to impart a hydrophobic property to a sugar chain, an aliphatic amine, sphingosine, phosphatidylethanolamine, or the like is bound via the reducing end of the sugar chain. Is also good. In the case of derivatives of glycoconjugates (glycopeptides), for example, a fatty acid, biotin, or the like is bound by utilizing the amino group or the N-terminal amino group of a lysine residue in the peptide moiety. Derivatives can be obtained.

[0096] In order to confirm the purity and structure of the sugar chains, glycoconjugates, derivatives thereof, and the like used in the present invention, in the case of sugar chains, the reducing terminal is a fluorescent substance such as 2-aminopyridine-pyrene hydrazine. The resulting labeled product was analyzed by HPLC or the like, and in the case of complex carbohydrates, for example, glycopeptides, the peptide portion was labeled with a fluorescent substance such as dansyl ore, FITC, etc. The yield and purity can be confirmed by analyzing the labeled product by HPLC or the like. In addition, such a labeled substance is also included in the derivative used in the present invention. Further, by determining the molecular weight by performing LC-MS analysis or TOF-MS analysis, the structures of the sugar chains, glycoconjugates, and derivatives thereof used in the present invention can be confirmed.

[0097] The sugar chains, glycoconjugates 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. Although not provided, for example, it is provided in the form of a salt such as a sodium salt, a potassium salt, a calcium salt, an ammonium salt, a chloride, and a carbonate. The salt of the sugar chain, the salt of the complex saccharide or the salt of a derivative thereof can be used, for example, for general desalting such as Dowex 50W, trade name: Dowex MR3 (both manufactured by Dow Chemical Company). It can be obtained as free sugar chains, complex carbohydrates or derivatives thereof by using ion-exchange resin, and is more likely to be exchanged with other salts.

[0098] The effect of inducing neurite outgrowth by the neurite outgrowth inducing agent of the present invention is, for example, nervous system cells, preferably, for example, neuroblastoma cells, pheochromocytoma cells, and glia. The cell tumor is evaluated by culturing the cell tumor cells in the presence of a sample to be tested, and observing the obtained cells. It is an indicator of having an induction effect. In addition, the effect on cell spreading can be evaluated by the same method.

[0099] Specific examples of the nervous system cells include human neuroblastoma cell line SK-, such as mouse neuroblastoma cell line Neuro-2a cell (ATCC CCL-131). C6, a rat astroglioma cell line, such as N-SH cells (ATCC HTB 11), SH-SY5Y cells, and rat chromaffin cell tumor cell line 1 ^ 12 cells (81-1 CRL1721) Cells (ATCC CCL-107) and the like.

[0100] The conditions for culturing the nerve cells are not particularly limited. 0.01% by weight of poly-L lysine in Dulbecco's modified Eagle's medium (DMEM) containing 10% by weight of sera and 5% by weight of FCS is used. Conditions include inoculating a coated culture dish into 1 × 10 ⁵ Z dishes and culturing at 37 ° C. in the presence of 5% by volume CO.

2

[0101] The concentration of the test sample in the medium can be appropriately set.

[0102] The formation of neurites in cells can be observed by, for example, a phase contrast microscope or the like. In the present invention, preferably, for example, a cell having two or more neurites of 3 m or more is a “cell having a neurite”, and a cell having low brightness when observed with a phase contrast microscope is “extended”. Cells ".

[0103] The neurite outgrowth inducing agent of the present invention may be evaluated for its ability to induce differentiation into cells. The differentiation-inducing ability is determined by culturing the nervous system cells in the presence of a sample to be tested, and examining the obtained cells by using various markers that serve as indicators of cell sorting, for example, Nippon Filament H or the like. It can be evaluated by measuring the presence or absence of expression.

The bisecting GlcNAc-containing sugar chains, glycoconjugates, derivatives thereof and Z or salts thereof (hereinafter sometimes referred to as the active ingredient of the present invention) used in the present invention have a neurite elongation activity. Neurite outgrowth agent (that is, neurite outgrowth inducer), and as a therapeutic or prophylactic agent effective for the treatment of neurological disorders, and as an enhancer or improver of learning and Z or memory ability. Can be. Said neurological disease means a disease accompanied by neurodegeneration, for example, a brain tumor (e.g., accompanied by nerve damage, deterioration of nerve function). Brain tumors); neurodegenerative diseases such as Aln's disease, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis; dementia; and diseases such as cerebral ischemic injury.

[0105] Furthermore, in the present invention, an enzyme involved in 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. Examples of the enzyme include, but are not limited to, GnT-ΠΙ and the like. Examples of the nucleic acid encoding GnT—ΙΠ include a nucleic acid encoding rat-derived GnT-III [Journal of Biological Chemistry, Vol. 267, pp. 18199 to 18204, 1992] and a human-derived GnT III. [Journal of Biochemistry, Vol. 113, pp. 692-698, 1992].

[0106] According to the GnT-III, a sugar chain having bisecting GlcNAc is synthesized in cells to which the GnT-III has been administered, thereby exerting an excellent effect of expressing a neurite elongation effect. Further, according to the nucleic acid encoding GnT-III, in a cell to which the nucleic acid encoding GnT-III is administered, the nucleic acid is translated into the nucleic acid GnT-III, and the sugar having bisecting GlcNAc is converted by the GnT-III. Chains are synthesized, thereby exerting an excellent effect of expressing a neurite elongation effect.

[0107] Therefore, in another aspect, the present invention relates to an agent for treating or preventing a neurological disease, an agent for treating or preventing dementia, an agent for improving and improving learning and Z or memory ability.

[0108] In still another aspect, the present invention relates to a method for producing a therapeutic or prophylactic agent for a neurological disease, a therapeutic or prophylactic agent for dementia, an agent for improving or improving learning and Z or memory ability, and the like. A sugar chain having the bisecting GlcNAc, in particular, a sugar chain represented by the formula (2), a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex saccharide, and the sugar At least one selected from the group consisting of a pharmacologically acceptable salt of a chain, 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 use of

[0109] The neurite can be elongated by the sugar chains, glycoconjugates, derivatives thereof and Z or salts thereof having the above-mentioned Neusecting GlcNAc, so that neurites can be elongated in individuals, for example, humans, non-human animals, etc. Treatment or prevention of neurological disorders, learning and improvement of z or memory ability or Can be improved.

[oiio] The therapeutic or preventive agent for neurological diseases, the therapeutic or preventive agent for dementia, the enhancer or improver for learning and Z or memory ability of the present invention are commonly used in injections, oral preparations, ointments and the like. hand

Although the preparation form can be used, it is preferably administered directly to the affected area as an injection. When prepared as an injection, a buffer solution such as PBS, a pH-adjusted preparation, physiological saline, a stabilizer, etc. are added to the sugar chain derivative of the present invention, and intravenous, intradermal, subcutaneous, and muscle An injection for intravenous administration can be prepared.

[0111] The neurite outgrowth inducing agent, the therapeutic or preventive agent for a neurological disease, the enhancer or improver for learning and Z or memory ability of the present invention comprises a sugar chain having bisecting GlcNAc, and the sugar chain in the structure. At least one selected from the group consisting of a complex carbohydrate, a sugar chain or a derivative of the complex carbohydrate, and a pharmacologically acceptable salt strength of the sugar chain or the complex carbohydrate as an active ingredient If it contains, the dosage varies depending on the patient (eg, body weight, age, medical history, etc.) and indications. For example, as an injection, the dose of the active ingredient is from 0.001 to the LOO mg. So, it is preferable to administer.

[0112] When containing the neurite outgrowth inducing agent of the present invention, a therapeutic or prophylactic agent for a neurological disease, an enhancer or improver of learning and Z or memory ability GnT-III as an active ingredient, to a cell Can be administered by a method used for known protein drugs, for example, by administering to a target cell or a tissue around the target cell as an injection.

[0113] The neurite outgrowth inducing agent, the therapeutic or preventive agent for a neurological disease, the enhancer or improver for learning and Z or memory ability of the present invention comprises a nucleic acid encoding GnT-III as an active ingredient. In some cases, administration to cells can be performed by known gene transfer methods.

[0114] Examples of the gene transfer method include a calcium phosphate method, a microinjection method; a physicochemical method such as an electoral poration method, a lipofection method, and a ribosome method; adenovirus, SV40 virus, and simple virus. Examples include a method using a virus vector such as a virus, an adeno-associated virus, a retrovirus, and a lentivirus. In the physical method, a DNA fragment containing the nucleic acid or a vector into which the nucleic acid is inserted, such as a plasmid vector, is introduced into cells. Hope for continuous effect In this case, it is preferable to use a virus vector having a property of integrating a target gene such as an adeno-associated virus, a retrovirus, or a lentivirus into a chromosome.

[0115] The neurite outgrowth agent of the present invention for a living body, a therapeutic or preventive agent for brain tumor 'neurodegenerative disease' dementia, cancer and malignant tumor, and an enhancer or improver for learning and Z or memory ability are As a DNA fragment containing a nucleic acid as an active ingredient, or a nucleic acid incorporated into an appropriate vector, it may be administered to a site where administration is desired, or cells into which the nucleic acid has been introduced may be implanted at a site where administration is desired. You can.

[0116] If GnT-III or a nucleic acid encoding GnT-III is administered to cells in the vicinity of cells where neurite outgrowth is desired (target cells), it may not be administered to target cells. The object of the present invention can be achieved because the neurite outgrowth effect is exerted even on the target cell having the sugar chain force having Neusecting GlcNAc generated in the cells surrounding the cell.

[0117] The therapeutic or preventive 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 It is preferable that 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 is contained as an active ingredient. There is a big feature. Therefore, according to the therapeutic or preventive agent for a neurological disease of the present invention, neurite outgrowth is induced in an individual suffering from a neurological disease, thereby exhibiting an excellent effect of being able to treat or prevent a neurological disease. I do.

[0118] The pharmacological evaluation of the therapeutic agent or prophylactic agent for a neurological disease of the present invention includes, for example, an evaluation method in an in-vivo mouth by the same method as the evaluation of the inducing effect of neurite outgrowth; an evaluation method using a neurological disease model animal ; Analysis by positron emission tomography in individuals with neurological disease; Measurement of brain function by magnetoencephalography in individuals with neurological disease; Functional analysis by drawing magnetic resonance function in individuals with neurological disease It can be. Evaluation methods using neurological disease model animals include, for example, administering a drug to be evaluated to model animals such as mice, rats, rabbits, dogs, monkeys, etc., and examining changes in disease-based physiological indices in the model animals. Diagnosing changes in Z or biochemical indicators, anatomy It is performed by observing a diseased part by a typical examination. Here, in the model animal, the physiological index and the Z or biochemical index are determined based on the physiology of a normal animal serving as a source of the neurological disease model animal (ie, an individual substantially not suffering from a neurological disease). In the case where the same condition as the clinical index and Z or biochemical index is shown, the disease state at the diseased site is the same as the corresponding site in the normal animal, etc. It is an indicator of having a therapeutic or preventive effect on the disease. In addition, analysis by positron emission tomography shows that, at the site of disease, for example, the turnover of neurotransmitters, such as donotamine, This can be done by analyzing the reuptake site and the like. Here, when the turnover or the like shows the same state as the corresponding site in a normal individual, it becomes an index that the drug to be evaluated has a therapeutic or preventive effect for a neurological disease.

[0119] The agent for improving or improving the learning and / or memory ability of the present invention is a sugar chain having bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, or a complex of the sugar chain or the complex saccharide. At least one selected from the group consisting of a derivative, a pharmacologically acceptable salt of the sugar chain or the glycoconjugate, an enzyme involved in synthesizing the sugar chain, and a nucleic acid encoding the enzyme as an active ingredient There is one major feature in its inclusion. Therefore, according to the agent for improving or improving the learning and Z or memory ability of the present invention, neurite outgrowth is induced in an individual in need of improving or improving the learning and Z or memory ability, and thereby the learning is improved. And Z or memory ability can be improved or improved.

[0120] In this specification, "improvement of learning and Z or memory ability" refers to the level of learning and Z or memory ability that is normally exerted by normal individuals, for example, humans and non-human animals. This means that the level is substantially increased. In addition, "improvement of learning and Z or memory ability" refers to an individual who has substantially lost learning and Z or memory ability due to a neurological disease, cerebral ischemic injury, trauma, etc., or has reduced learning and Z or memory ability. In an individual is meant to substantially increase the level of learning and Z or memory capacity.

[0121] The pharmacological evaluation of the learning and Z or memory capacity improving agent or the improving agent of the present invention can be carried out by, for example, an evaluation method using a normal animal, a learning and Z or model animal with low memory capacity, a learning ability, It can be performed by a force test, a memory test, and the like. The evaluation method using the normal animal, the model animal, and the like is, for example, to administer a drug of a test subject to an animal such as a mouse, a rat, a penguin, a dog, or a monkey, and get out of a maze where a bait is placed at a goal point. This can be done by measuring the time several times. Here, as compared with a normal animal to which the test subject drug is not administered, in a normal animal to which the test subject drug is administered, the starting point force also has a greater reduction in the time required to reach the goal point where the food is placed. The drug of the subject is learning and

It is an index to show improvement of Z or memory ability. In addition, compared to the model animal before administration of the test subject drug, the time required from the start point to the goal point where the food was placed in the model animal to which the test subject drug was administered was shortened. It is an indicator that the subject's drug exhibits improved learning and Z or memory ability.

[0122] In another aspect, the present invention relates to a method for inducing neurite outgrowth.

The method for inducing neurite outgrowth of the present invention comprises a sugar chain having bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex saccharide, and the sugar chain. At least one selected from the group consisting of a pharmacologically acceptable salt of the above, a pharmacologically acceptable salt of the glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. One feature is that it is administered to the specimen.

[0124] Examples of the specimen include cultured cells, nerve tissue, and animal individuals. Examples of the animal individual include a human and a non-human animal.

[0125] The effect of inducing neurite outgrowth by the induction method of the present invention can be evaluated by the same method as the method of evaluating the effect of inducing neurite outgrowth by the above neurite outgrowth inducer.

[0126] In the induction method of the present invention, administration can be carried out in the same manner as in the case of the aforementioned 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.

[0127] Cultured cells (eg, cells of the nervous system, neural progenitor cells) and nerve tissue in which neurites have been extended by the induction method of the present invention can be used for transplantation for the treatment of neurological diseases.

[0128] In another aspect, the present invention relates to a method for treating or preventing a neurological disease.

[0129] The method for treating or preventing a neurological disease according to the present invention provides an individual having a neurological disease or the neurological disease. A sugar chain having the bisecting GlcNAc, particularly a sugar chain having the bisecting GlcNAc represented by the above formula (2), is provided to an individual who is likely to suffer from the disease. 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 the complex carbohydrate, an enzyme involved in the synthesis of the sugar chain, and One feature is that at least one selected from the group consisting of nucleic acids encoding the enzyme is administered.

[0130] Examples of the individual include a human and a non-human animal.

[0131] The therapeutic or preventive effect of the method for treating or preventing a neurological disease of the present invention can be evaluated by the same method as the pharmacological evaluation of the therapeutic or prophylactic agent for a neurological disease of the present invention.

[0132] In the method for treating or preventing a neurological disease of the present invention, administration can be performed in the same manner as in the case of the therapeutic or prophylactic agent for a neurological disease of the present invention. In the method for treating or preventing a neurological disease of the present invention, the therapeutic or prophylactic agent for a neurological disease of the present invention may be used.

[0133] In still another aspect, the present invention relates to a method for improving or improving learning and Z or memory ability.

[0134] The method for improving or improving the learning and Z or memory ability of the present invention can be applied to an individual in need of improving or improving the learning ability for the sugar chain having the bisecting GlcNAc, in particular, the formula (2) A sugar chain having the bisecting GlcNAc represented by the formula, a complex carbohydrate having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, and a pharmacologically acceptable sugar chain Providing at least one member selected from the group consisting of a pharmaceutically acceptable salt, a pharmacologically acceptable salt of the glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Great features.

[0135] The individual in need of improving or improving the learning ability includes a normal individual, for example, a human, a non-human animal, or the like; and has a substantial learning and Z or memory ability due to a neurological disease, cerebral ischemic injury, trauma, or the like. Individuals who have lost learning and Z or memory ability due to neurological diseases, cerebral ischemic injury, trauma, and the like.

[0136] The learning and Z or memory capacity improvement or improvement effect of the learning and Z or memory capacity improvement or improvement method of the present invention is based on the learning and Z or memory capacity improver or modifier. It can be evaluated by the same method as the pharmacological evaluation of a good drug.

[0137] In the method for improving or improving the learning and Z or memory ability of the present invention, administration can be performed by the same method as that for the therapeutic or preventive agent for a neurological disease of the present invention. In the method for improving or improving the learning and Z or memory ability of the present invention, the agent for improving or improving the learning and Z or memory ability of the present invention can be used.

[0138] In another aspect, the present invention relates to a food or beverage.

The food or beverage of the present invention includes a sugar chain having bisecting GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex saccharide, and pharmacology of the sugar chain. One major feature is that it contains at least one (the above-mentioned active ingredient) selected from the group consisting of specifically acceptable salts and pharmacologically acceptable salts of the glycoconjugates. Therefore, the food or beverage of the present invention is extremely useful for ameliorating and preventing the symptoms of the above-mentioned diseases due to its neurite outgrowth action. Further, the food or beverage may be a functional food or a functional beverage (for example, a food for specified health use) indicating that the purpose is to improve or prevent the symptoms of the above-mentioned diseases.

[0140] The food or beverage of the present invention contains the active ingredient, and thus can be used for treating or preventing a neurodegenerative disease, dementia, or brain tumor. Further, since the food or beverage of the present invention contains the active ingredient, it can be used for improving or preventing learning and Z or memory ability. The food or beverage of the present invention includes food or beverage for assisting treatment or prevention of neurodegenerative disease, dementia or brain tumor, and food or beverage for assisting improvement or prevention of learning and Z or memory ability. Beverages are included.

[0141] Here, the term "containing" used for the food or beverage of the present invention includes the meaning of "addition" and / or "dilution".

[0142] In the present specification, the term "containing" used for the food or beverage of the present invention refers to an embodiment in which the active ingredient used in the present invention is contained in a food or beverage, and the term "added" The term `` dilute '' refers to adding the active ingredient used in the present invention to the raw material of food or beverage, and the term `` dilution '' refers to adding the raw material of food or beverage to the active ingredient used in the present invention. Then, the mode is changed. [0143] The method for producing a food or beverage of the present invention is not particularly limited. For example, blending, cooking, processing, etc. can be carried out according to general food or beverage production methods, and can be produced by such production methods.If the obtained food or beverage contains the active ingredient, Just fine.

[0144] The food or beverage of the present invention is not particularly limited as long as it contains the above-mentioned active ingredient. Examples thereof include processed cereals (processed flour, processed starches, premixed cascine). Products, vegetables, macaroni, bread, bean jam, buckwheat, fu, rice noodles, harame, packing mochi, etc., processed oils and fats (plastic oils, tempura oil, salad oil, mayonnaise, dressing, etc.) , Processed soybeans (tofu, miso, natto, etc.), processed meats (ham, bacon, pressed ham, sausage, etc.), marine products (frozen surimi, rikamaboko, chikuwa, hampon, satsumaage, tsumire, Streaks, fish ham, sausage, bonito, processed fish eggs, canned fish, boiled tsukudani, etc.), dairy products (raw milk, cream, yogurt, butter, cheese, condensed milk, milk powder, ice cream, etc.), processed vegetables and fruit (Pastes, jams, pickles, fruit drinks, vegetable drinks, mixed drinks, etc.), confectionery (gum, candy, chocolate, biscuits, confectionery breads, cakes, rice cakes, rice crackers, etc.), alcoholic beverages (sake) , Chinese sake, wine, whiskey, shochu, vodka, brandy, gin, rum, beer, soft alcoholic beverages, fruit liquor, liqueurs, etc., favorite beverages (green tea, black tea, oolong tea, coffee, soft drinks, lactic acid drinks) Etc.), seasonings (soy sauce, sauce, vinegar, mirin, etc.), canned 'bottled' bagged foods (beef rice, pot rice, red rice, curry, and various other prepared foods), semi-dried or concentrated foods (liver paste, Other spreads, soba noodle soup, concentrated soups, dried food (instant types, instant curry, instant coffee, powdered juice, powdered soup, instant miso soup) Prepared foods, cooked beverages, cooked soups, etc.), frozen foods (sukiyaki, chawanmushi, eel kabayaki, hamburger steak, shiyumai, gyoza, various sticks, fruit-based kettle, etc.), solid foods, liquid foods (soups, etc.) And agricultural products such as spices, processed forest products, processed livestock products, processed marine products, and the like.

[0145] The food or beverage of the present invention contains, adds and Z or is diluted with the above-mentioned active ingredient singly or as a mixture of plural kinds thereof, and contains a necessary amount for expressing a neurite elongation effect. Orally ingestible tablets, granules, capsules, etc. Also includes various shapes.

[0146] 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 elongation activity. For example, in the food or beverage of the present invention, the content is 0.01% by weight or more. Preferably, it is at least 0.1% by weight, more preferably at least 1% by weight, at most 10% by weight, more preferably at most 5% by weight. The content can be arbitrarily selected within the above range. Further, the food or beverage of the present invention preferably contains an active ingredient contained therein in an amount of 0. OOOOlmg ~: LOOOmgZ kg body weight per day, preferably <0. OOOlmg ~: LOOmgZkg body weight per human (for example, adult). , More preferred <0. OOlmg ~: LO mgZkg should be ingested so that the body weight intake. Such an intake may fluctuate depending on various conditions, and in some cases, an amount smaller than the above-mentioned intake may be sufficient, or in other cases it may be necessary to exceed the range.

[0147] The food or beverage of the present invention can be evaluated, for example, by an evaluation method using a neurological model animal used for the pharmacological evaluation of the therapeutic or prophylactic agent for a neurological disease of the present invention.

[0148] By the way, a sugar chain having a GlcNAc residue at the non-reducing end and having a three- or four-chain structure, or a complex saccharide having the sugar chain, which is produced by the action of GnT-V, However, as is apparent from the suppression of wound closure described in the Examples below, it has an activity of inhibiting cell migration and movement, and is therefore effective in treating malignant tumors by inhibiting cancer cell invasion and metastasis. It can be used as a therapeutic or metastasis inhibitor. The above-mentioned therapeutic agent or metastasis inhibitor for malignant tumor can be produced according to the therapeutic or preventive agent for neurological diseases of the present invention.

[0149] Power for explaining the present invention more specifically by the following examples and the like The present invention is not limited to only such examples.

Example 1

Production of Gn (Gn) Gn-bi-glycopeptide

(1) Preparation of -Avian egg yolk power sialyl glycopeptide (SGP)

An equal volume of water was added to 1 L (for 50) of white leghorn fresh egg yolk. To the obtained egg yolk dilution, 1Z10 amount of phenol Z water (9: 1, weight ratio) was added, and the mixture was vigorously stirred for 2 hours. 7000 The mixture was centrifuged at X 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: Sep hadex G-50 (manufactured by Amersham Bioscience) column (2.5 cm × 100 cm). At this time, 0.1 M NaCl was used as an eluent. The sugar contained in the eluted fraction was analyzed using the phenol-sulfuric acid method (edited by the Biochemical Society of Japan, New Chemistry Laboratory Course, Vol. 3, Carbohydrate I, Glycoprotein (above), p. ), And the second major eluted sugar-positive fraction was collected. The sugar-positive fraction was concentrated to 5 mL using a rotary evaporator. The obtained product was desalted using a Sephadex G-25 (manufactured by Amersham Bioscience) column (2.5 cm × 52 cm). At this time, 5% by volume of ethanol was used as an eluent. The sugar-positive fractions were collected by measuring the sugar content of each fraction by the phenol sulfate method. The obtained product was concentrated to 5 mL with a rotary evaporator to obtain a concentrated solution. 5 mM Tris-HCl (pH 8.0) equilibrated Trade name: TOYOPEARL DEAE-650M (manufactured by Tosoichi Co., Ltd., product number: 07473) Fractions were collected. The flow-through fraction was concentrated to 5 mL using a rotary evaporator. The obtained product was desalted using a Sephadex G-25 column (trade name) and lyophilized to obtain SGP.

(2) Removal of sialic acid and galactose from SGP by enzyme treatment

Example 1—The SGP obtained in (1) was dissolved in water so as to be 170 / z mol Zl. 5 mL. To the obtained solution, 45 mg of powder EZ-Link Sulfo-NHS-Biotin (trade name, manufactured by PIERCE, product number: 21217) was added, and the mixture was vigorously stirred immediately and then shaken at room temperature for 1 hour. The obtained mixture was concentrated to 0.5 mL using a centrifugal concentrator. To the obtained concentrate, 2.4 mL of lOOmM sodium citrate buffer (pH 5.0) and 3UZ300 L of neuraminidase (manufactured by Nacalai Tester, product number: 24229-74) were added, and the mixture was added at 37 ° C for 24 hours. Reacted. To the obtained product, 3UZ70 / ZL | 8-galactosidase (manufactured by Seikagaku Corporation, product number: 100570) was added, 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 13,000 × g (14000 rpm) for 10 minutes. The obtained supernatant was concentrated to 400 L using a centrifugal concentrator. The obtained product is used as a PD-10 desalting column (Amersham Bioscience) Desalinated by the company, product number: 17-0851-01), further loaded onto a cellulose cartridge column (manufactured by Takara Bio Inc., product number: 4404), purified, and crudely purified biotinylated. Gn.Gn-bi-glycopeptide (hereinafter, “crudely purified Gn.Gn-bi-GP”) was obtained. The structure of Gn.Gn-bi-GP (SEQ ID NO: 1) is shown in the following formula (9).

[0152] [Formula 24]

Lys (NH ₂ )

GlcNAc i-2Manal \ Val

6 Ala

Manpi-4GlcNAcpi-4 GlcNAcpl-Asn

3 Lys

GlcNAcpi-2Manar Thr (COOH)

(9)

(3) Introduction of bisecting GlcNAc residue by GnT-III

Rat GnT-III cDNA [-Nishikawa, A., et al., Journal of Biological Chemistry, Vol. 267, pp. 18199-18182, 1992] was converted to pCXNII [-ヮ (Niwa) et al., Gene, Vol. 15, pp. 193-199, 1991] to construct the plasmid pCXNIl / GnT-III.

Trade name: Lipofectamine 2000 (manufactured by Invitrogen, trade number: 11668-019) was used to transfer the above pCXNIlZGnT-m to rat adrenal pheochromocytoma cell line PC12 (ATCC CRL-1721). Transfat, 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% by weight ゥ serum and 5% by weight ゥ fetal serum (FCS) and lmgZmL G418

2. Cultured at 37 ° C. After 2 weeks of culture, colonies were isolated and recloned by the limiting dilution method 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 put on three collagen-coated 10 cm dishes (manufactured by Asahi Techno Glass Co., Ltd., product number: 4020-010) and contained 10% by weight of serum and 5% by weight of FCS. Culture in DMEM in the presence of 5% by volume CO at 37 ° C until confluent

2

7

[0155] After completion of the culture, the medium was discarded and washed with phosphate buffered saline [PBS (-)]. Separate the cells from the dish using a cell scraper and centrifuge at 1500 X g (5000 rpm) for 5 minutes. Separated and cells harvested. The supernatant is discarded, and the obtained cells are supplemented with 200 μL of PBS (-) containing 10 μg mL of leupeptin, 10 / zg / mL aprotune, and ImM fluoride methyl sulfol. The mixture was polished 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 L of a supernatant. The resulting supernatant was used as a GnT-III crude enzyme solution.

[0156] 100 µL of GnT-ΙΠ crude enzyme solution and 200 µL of 2X buffer [composition: 250 mM MES-NaOH (pH 6.25), 20 mM MnCl, 400 mM N-acetyldarcosamine (GlcNA

2

c), 1.0% (V / V) Triton ™ X-100], 40 L of 500 mM UDP-GlcNAc, and 50 μL of 0.825 μmol / 50 prepared in Example 1- (2) μL The crudely purified aqueous solution of Gn.Gn-bi-GP was mixed with 10 / z L of water, 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 X g (14000 rpm) for 10 minutes to obtain a supernatant. Trade name: The supernatant was desalted using a PD-10 column, purified using a cellulose cartridge column, concentrated under reduced pressure, and evaporated to dryness. The obtained product was dissolved in 200 / zL of water to obtain a sample A containing crude purified Gn (Gn) Gn-bi-glycopeptide. The glycopeptide concentration in sample A was 4. ImM when measured by the phenol sulfate method. The structure of Gn (Gn) Gn-bi-glycopeptide (SEQ ID NO: 1) is shown in the following formula (10).

[0157] [Formula 25]

Lys (NH ₂ )

GlcNAc l-2Man l \ Val

6 Ala

GlcNAc i-4 Man i-4GlcNAcpl-4 GlcNAcpi-Asn

Lys

GlcNAcpl-2Manar Thr (COOH)

(Ten)

(4) Analysis of sugar chain structure of sample A

The sugar chain structure of Sample A was analyzed by the following method. Sample A was diluted 100 times with water. 1) 0.5 M of 0.5M sodium phosphate buffer (pH 7.5) and 0.5 / zL of 10% by volume Nonidet (trade name) PZ40 (NP — 40, Merck, product number: BDH560092L) and 0.5 L of 500 UZ μL peptide: Ν—Glycosidase F (PNGase F, New England BioLabs, product number: P0704S) , twenty five / zL of water was added and reacted at 37 ° C for 24 hours. The resulting reaction solution was boiled for 5 minutes, and centrifuged at 130,00 × g (14000 rpm) for 5 minutes to obtain a supernatant.

The total amount of the supernatant was subjected to pyridylamino (PAM-Doll, trade name: GlycoTAG Reagent Kit (manufactured by Takara Bio Inc., trade number: GT500)). And 20 μL thereof was analyzed by HPLC.In the HPLC, TSKgel ODS-80T (4.6 × 150 mm, Tosoh Corporation) was used as a column.

M

, Product number: 08148), 20 mM ammonium acetate buffer containing 0.2% by volume of 1-butanol (pH 4.0) as eluent, column temperature 50 ° C, flow rate 1. It was set to 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.

[0160] As a result, no PA-Gn (Gn) Gn-bi-sugar chain was detected from the crudely purified Gn.Gn-bi-GP, whereas 15% of PA-Gn ( Gn) Gn-W-sugar chain was detected.

Example 2

[0161] Production of Gn.Gn.Gn-tri'-glycopeptide

(1) Introduction of j8 1, 6-GlcNAc residue into biotinylated Gn.Gn-bi-glycopeptide

According to the method of Gu et al. (Gu, J., et al., Journal of Biochemistry, Vol. 113, pp. 614-619, 1993), human small cell lung cancer cell line QG was also purified from GnT-V. . Using the obtained GnT-V, the same reaction as in Example 1- (3) was performed on crude Gn.Gn-W-GP to obtain a sample B. Incidentally, when the reaction using GnT- V, as a buffer, 2 X buffer solution [composition: 250mM MES -NaOH (. PH6 25 ), 400mM GlcNAc, 20mM E DTA '3Na, 1. 0 volume ^_0/0 Triton ™ X-100] was used.

[0162] The structure of the product Gn.Gn.Gn-tri'-glycopeptide (SEQ ID NO: 1) is shown in the following formula (11). [0163] [Formula 26]

Manpl-4GlcNAcpi-4 GlcNAcpi-Asn

'' Lys

GlcNAcpi-2Manal 'Thr (COOH)

(2) Analysis of sugar chain structure of sample B

Under the same conditions as in Example 1 (3), the sample B was analyzed by PNGase F treatment, PA gel and HPLC. Under these conditions, the PA-Gn.Gn-bi-glycan eluted at 7.1 minutes and the PA-Gn.Gn.Gn-tri'-glycan eluted at 5.2 minutes.

[0165] As a result, PA-Gn.Gn.Gn-tri'-sugar chain was not detected from the crudely purified Gn.Gn-bi-GP, whereas 63% of PA-Gn was detected from sample B. .Gn.Gn-tri'-sugar chains were detected.

Example 3

[0166] Neurite formation and cell spreading by Gn (Gn) Gn-bi-glycopeptide and Gn.Gn.Gn-tri'-glycopeptide

(1) Neurite formation and cell spreading of Neuro-2a cells by Gn (Gn) Gn-bi-glycopeptide and Gn.Gn.Gn-tri'-glycopeptide

Example 1—200 μL of GnT-ΠΙ crude enzyme solution prepared by the method of (3) was heat-treated at 98 ° C for 5 minutes, and centrifuged at 13000 X g (14000 rpm) for 10 minutes to obtain a supernatant. Was. The obtained supernatant was loaded on a cellulose cartridge column (manufactured by Takara Bio Inc., product number: 4404) to obtain an eluted fraction. This fraction was concentrated by centrifugation to dryness, and dissolved in 200 L of water to obtain Sample C.

[0167] Sample D was obtained from PC 12 (mock) cells by the same method.

[0168] A mouse neuroblastoma cell line Neuro-2a (ATCC CCL-131) was suspended in DMEM containing 10% by weight of serum and 5% by weight of FCS, and 0.01% poly-L-lysine was added. 1 x 10 ⁵ pieces of 35 mm glass bottom culture dish (MatTek, product number: P35G—0—10—C) coated with Sigma (product number: P4832) It was inoculated so that Itsushu, 5 volumes ^_0/0 CO presence and 1晚incubated at 37 ° C. After the medium was removed by suction, 100 L of a diluted sample obtained by diluting Sample A, Sample C or Sample D 100-fold with lOmM HEPES-containing PBS (-) was added to the cavity of the dish. Incubated at 90 ° C for 90 minutes. The cells were then fixed with 3.7% by volume formalin in PBS (-). The fixed cells were observed with a phase-contrast microscope (Carl Zeiss, trade name: Axiovert 200M), and the ratio of cells having neurites and the ratio of expanded cells were calculated. Here, cells with two or more neurites of 3 m or more are called “cells with neurites”, and cells with low brightness when observed with a phase contrast microscope are called “extended cells”.

[0170] The results are shown in Fig. 1. FIG. 1 is a diagram 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 visual fields. Most of the cells showed neurite outgrowth and cell outgrowth in the sample A-added calo section. In contrast, in the sample C-added group, slight neurite formation was observed, but no cell extension was observed.In the sample D-added group, neurite formation and cell extension were hardly observed. Helped.

[0171] Using a roughly purified Gn.Gn-W-GP, sample A and sample B as test samples, cells were incubated in the same manner as described above, and formalin fixation was performed 90 minutes after the addition of the test sample. After formalin fixation, the cells were washed with PBS (-). Then, actin was stained with Alexa 546-Phalloidin, and observed with a confocal laser microscope (manufactured by Carl Zeiss, product number: LSM 5 Pasca 1).

[0172] As a result, most of the cells were spherical and almost no neurites were found in the roughly purified Gn.Gn-bi-GP-supplemented kaju, while Gn (Gn) Gn-bi In the sample containing A-GP, most cells formed many neurites and showed cell spreading. In addition, in the sample B-added group containing Gn.Gn.Gn-tri'-GP, most cells were spherical and almost no neurite formation was observed, but phytopodia-like actin cytoskeleton reorganization was observed. Was done.

[0173] (2) Induction of Neuro-2a cell differentiation by Gn (Gn) Gn-bi-glycopeptide

After culturing Neuro-2a cells in the same manner as in Example 3 (1), remove the medium, and add 2 mL of sample A, DMEM or DMEM containing 10% by weight of FCS diluted to 100 times with DMEM, and add 5 mL of caloric solution. The cells were cultured at 37 ° C for 36 hours in the presence of% CO. Remove the medium and add 4%

2

The cells were fixed with formaldehyde-containing PBS for 1.5 minutes. Then 1% by weight Triton ™ The mixture was gently shaken at room temperature for 15 minutes in 50 mM Tris-HCl (pH 6.8) containing X-100. Cells were fixed again with 4% by weight paraformaldehyde for 30 minutes at 4 ° C. Cells were washed with PBS and incubated in 50 mM NH Cl for 30 minutes. Next, the cells are

Four

Weight ^_0/0 © shea serum albumin 0.1 anti-phosphorylated was diluted 2000-fold with PBS containing 1 wt ^{_{0/0 Triton ™ X-100}} - Euro filaments H (NF-H) mouse monoclonal antibody SM 1-31 [ The cells were incubated at 4 ° C for 1 晚 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 of goat serum, and then incubated at room temperature for 2 hours in Alexa 546-labeled goat anti-mouse IgG diluted 300-fold with PBS. In addition, each incubation was washed 2-3 times with PBS. The cells after incubation were observed with a confocal laser microscope.

[0174] As a result, formation of neurites was scarcely observed in the kamuji section supplemented with 10% by weight of FCS and DMEM, and NF-H positive neurites were observed in some cells in the kamum section supplemented with DMEM. More NF-H positive neurites were observed in the sample A-added group than in the DMEM-added group.

(3) Expansion of T98G cells by Gn (Gn) Gn-bi-glycopeptide

The same experiment as in Example 3- (1) was performed using human glioma cells T98G (ATCC CRL-1690). However, crudely purified Gn.Gn-b-to-GP, sample A and sample B were used as test samples, and the dilution with PBS (-) containing 10 mM HEPES was made 50-fold. The time from sample addition to formalin fixation was 90 minutes, and observation by a phase-contrast microscope and observation of actin by Alexa 546-phalloidin (Invitrogen) staining were performed.

[0176] As a result, cell growth was inhibited in the roughly purified Gn. Partial cells spread and formed stress fibers prominently.

Example 4

[0177] Inhibition of T98G motility by Gn.Gn.Gn-tri'-glycopeptide

1 Ueru per 0. 5 X 10 ⁶ cells of T98G cells were seeded in 24-well plates, 10 weight ^_0/0 FC S-containing RPMI1640 medium, 5 volume% CO presence and 1晚incubated at 37 ° C. Bruch Using a tip, a line was drawn in the center of the well to make a wound of about 120 m in the cell layer, and the medium was carefully removed. After washing with DMEM containing 1% FCS, 250 L of the above medium, crude Gn. Gn-bi-GP diluted 500 times with the above medium, or sample B diluted 500 times with the above medium were added. After 24 hours incubation, 4.0 volume ^_0/0 paraformaldehyde containing PBS (-) were fixed with and observed with a phase contrast microscope.

[0178] As a result, when only the medium was added, the wound closure was enhanced and V (the wound in the cell layer was almost completely filled with cells), whereas Wound closure was suppressed in the crudely purified Gn.Gn-W-GP-added section, and further suppressed in the sample B-containing section containing Gn.Gn.Gn-tri'-glycopeptide.

Example 5

[0179] GnT-III gene transfer enhances neurite outgrowth

Rat GnT-III cDNA [-Nishikawa, A., et al., Journal of Biological Chemistry, Vol. . The obtained plasmid was introduced into Neuro-2A cells using Ribofectamine 2000 (manufactured by Invitrogen). Thereafter, transformed cells were selected using G418 resistance as an index, and multiple cell lines stably overexpressing GnT-III were established.

[0180] As a control, a cell strain (referred to as Mock) into which a plasmid plasmid pcDNA3.1 containing no GnT-ΠΙ cDNA was introduced was established in the same manner.

[0181] The same number of GnT-III transfected cell lines and Mock cell lines were each seeded on a 35 mm glass bottom culture dish so as to obtain a low density, and high-gluose tvpe containing 10% by weight of FCS was seeded. in DMEM medium (manufactured by SIGMA), and 12 hours at 5 volume ^_0/0 CO.

2

[0182] Thereafter, differentiation was induced by serum starvation by replacing the medium with a DMEM medium not containing FCS. As a control, a group that continued to culture both cell lines in a medium containing FCS was also tested. After culturing for 24 hours, the cells were fixed with PBS containing 4% by volume of formaldehyde for 10 minutes. After fixation, the cells were stained for the actin cytoskeleton, Alexa 546—Phalloidin, and for neuronal differentiation marker NF—H, anti-phosphorylated NF—H mouse monoclonal antibody SMI-31 and Alexa 546-labeled goat anti-mouse IgG antibody. For general use Was stained by a common indirect fluorescent antibody staining method and imaged with a confocal laser microscope.

[0183] The total number of cells and the number of NF-H-positive cells in the laser microscope visual field (73.1 x 73 .: Lm) were counted, and NF-H-positive (extended protruding part was detected with anti-NF-H antibody). Of the total cells was evaluated as a percentage. Three different clones were used in the experiment for each of the GnT-III gene-transfected cell line and Mock, and the average thereof is shown in FIG. In the figure, + and 1 indicate the presence or absence of FCS, respectively.

[0184] As a result, as shown in Fig. 2, it was revealed that the neurite outgrowth of Neuro-2a cell force accompanied by induction of serum starvation was enhanced by overexpression of GnT-III gene. Was.

[0185] The above results strongly suggest that the GnT-III gene product is deeply involved in the process of neurite outgrowth, particularly in neural differentiation.

Example 6

[0186] The glycopeptide (neurite outgrowth inducing agent) obtained in Example 2 was mixed under sterile conditions with a solution containing sterile physiological saline and an auxiliary for injection, and the mixture was injected into an injection formulation. To obtain a therapeutic or preventive agent for neurological diseases.

[0187] The therapeutic or preventive agent for a neurological disease is administered to a nerve injury site of an individual. Over time, the motor and speech abilities impaired by the nerve injury and the degree of recovery of response to Z or external force stimuli are assessed.

[0188] As a result, when the motor ability, speech ability impaired due to nerve damage, and the response to Z or external stimulus are restored, an index indicating that the pharmacological effect of the therapeutic or preventive agent for neurological disease was obtained. Is evaluated as

Example 7

[0189] The glycopeptide (a neurite outgrowth inducing agent) obtained in Example 2 is mixed with raw materials at the time of producing beverages and foods.

[0190] The obtained beverage or food is supplied to normal individuals and individuals whose learning ability and Z or memory ability have been reduced due to nerve damage. After that, they will test their learning and memory skills before and after serving the beverage or food and evaluate their performance.

[0191] Thus, compared to before the supply of the beverage or food, after the supply of the beverage or food, the normal individual Improved performance is an indicator that beverages or foods can improve learning and memory skills. In addition, if the performance of individuals with reduced learning ability and Z or memory ability due to nerve injury after the supply of beverages or foods approaches the normal level compared to before the supply of beverages or foods, the learning ability with the beverages or foods It is an indicator of the effect of improving memory ability.

Industrial applicability

According to the present invention, a sugar chain having Neusecting GlcNAc, a glycoconjugate, a derivative thereof, a salt thereof, an enzyme relating to the synthesis of the sugar chain, and a medicament or a reagent containing a nucleic acid encoding the enzyme , Food or beverage is provided. The drug, food or beverage is a neurite outgrowth-inducing effect of sugar chains, glycoconjugates, their derivatives, or Z or their salts having bisecting GlcNAc, thereby treating or preventing neurological disorders, and improving or improving learning and memory ability. Useful for Further, the present invention provides a reagent containing the active ingredient and having a neurite outgrowth-inducing action.

Sequence listing free text

[0193] SEQ ID NO: 1 shows the amino acid sequence of the peptide portion of a glycopeptide.

Claims

The scope of the claims

[1] bisecting GlcNAc-containing sugar chain, complex saccharide having the sugar chain in its structure, derivative of the sugar chain, derivative of the complex saccharide, pharmacologically acceptable salt of the sugar chain, A neurite elongation comprising as an active ingredient at least one selected from the group consisting of a pharmacologically acceptable salt of a glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Inducer.

[2] Neusecting Sugar chain with GlcNAc Formula (1) below:

[Formula 1] Sat GlcNAcp 2Manal \

6

GlcNAcpi-4Manpl- R ¹ (1)

Three

Sat GlcNAcpi-ZManct:

(In the formula, R ¹ represents a sugar residue, and 士 means the presence or absence of a GlcNAc residue.)

2. The neurite outgrowth inducing agent according to claim 1, which is a sugar chain represented by the formula:

[3] Neusecting Sugar chain force with GlcNAc The following formula (2):

[Formula 2]

R ² -Manal \

6

GlcNAcpi-4Manpi-R ^: (2)

Three

R ³ -Manal '

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are linked to mannose via a j8 1-2 bond, | 8 1-4 bond or | 8 1-6 bond. Indicates a GlcNAc residue (however, when R ² and R 3 have a GlcNAc residue, one or two R ² and R ³ may be directly bonded to mannose)]

[4] bisecting sugar chain having GlcNAc, complex saccharide having the sugar chain in its structure, derivative of the sugar chain, derivative of the complex saccharide, pharmacologically acceptable salt of the sugar chain, A neurological disease comprising as an active ingredient at least one selected from the group consisting of a pharmacologically acceptable salt of a glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. A therapeutic or prophylactic agent.

[5] Neusecting Sugar chain power with GlcNAc The following formula (1):

[Formula 3]

± GlcNAcpl-2Mana

GlcNAc i ^ Manpi-R ¹ (1)

Three

Sat GlcNAcpl-2Manal '

5. The therapeutic or preventive agent for a neurological disease according to claim 4, which is a sugar chain represented by the formula:

[6] Neusecting Sugar chain force with GlcNAc The following formula (2):

[Formula 4]

[7] The therapeutic or preventive agent for a neurological disease according to claim 1, which is used for treating or preventing a neurodegenerative disease, a dementia or a brain tumor.

[8] Bisecting A sugar chain having GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, a pharmacologically acceptable salt of the sugar chain, Learning and Z-containing at least one selected from the group consisting of a pharmacologically acceptable salt of a glycoconjugate, an enzyme involved in the synthesis of the sugar chain, and a nucleic acid encoding the enzyme. Or a memory capacity improver or improver.

[9] Neusecting Sugar chain with GlcNAc Formula (1) below:

[Formula 5] Sat GlcNAcpl-2M Marauder 1 \

GlcNAcpi- ^ Manpl-R ¹ (1)

Three

Sat GlcNAcpi-ZMana ^

(In the formula, R ¹ represents a sugar residue, and the symbol means presence or absence of a GlcNAc residue.)

9. The agent for improving or improving learning and Z or memory ability according to claim 8, which is a sugar chain represented by the formula:

[10] Neusecting Sugar chain with GlcNAc Formula (2) below:

[Formula 6]

GIcNAcpl-4Manpl-Ri ( ₂ )

Three

R ³ -Manal '

[11] Bisecting Sugar chain having GlcNAc, complex saccharide having the sugar chain in its structure, derivative of the sugar chain, derivative of the complex saccharide, pharmacologically acceptable salt of the sugar chain, and A food or beverage comprising at least one member selected from the group consisting of pharmacologically acceptable salts of glycoconjugates.

[12] Neusecting Sugar chain with GlcNAc Formula (1) below:

[Formula 7]

GlcNAcpi-2Manal \

D

GlcNAc l ^ Manpi-R ¹ (1)

Three

GlcNAcpi Manal ⁷

(In the formula, R ¹ represents a sugar residue, and the symbol means presence or absence of a GlcNAc residue.) 12. The food or beverage according to claim 11, which is a sugar chain represented by the formula:

[13] Neusecting Sugar chain force with GlcNAc The following formula (2):

[Formula 8]

R ² -Manal \

o

GlcNAcpl-4Manpi-R ^] (2)

Three

R ³ -Manotl,

12. The food or beverage according to claim 11, which is a sugar chain represented by the formula:

[14] The method according to claims 11 to 13, which is used for treating or preventing a neurodegenerative disease, dementia or brain tumor.

V, the food or beverage according to item 1.

[15] The food or beverage according to any one of claims 11 to 13, which is used for improving or preventing learning and Z or memory ability.

[16] Bisecting A sugar chain having GlcNAc, a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, a pharmacologically acceptable salt of the sugar chain, Administering at least one selected from the group consisting of a pharmacologically acceptable salt of a glycoconjugate, an enzyme involved in synthesizing the sugar chain, and a nucleic acid encoding the enzyme to a specimen. A method for inducing protrusion elongation.

[17] Sugar chain having Neusecting GlcNAc The sugar chain having bisecting GlcNAc is represented by the following formula (1):

[Chem. 9] Shido

(In the formula, R ¹ represents a sugar residue, and 士 means the presence or absence of a GlcNAc residue.) 17. The method for inducing neurite outgrowth according to claim 16, which is a sugar chain represented by the formula:

[18] Neusecting Glycoside power with GlcNAc The following formula (2):

[Formula 10]

17. The method for inducing neurite outgrowth according to claim 16, which is a sugar chain represented by the formula:

[19] The following formula (2) is given to an individual suffering from a neurological disease or an individual at risk of suffering from the neurological disease.

[Formula 11]

R ² -Manal \

D

GlcNAc l-4Manpl-R ¹ (2)

Three

R'-Manal

A sugar chain having a bisecting GlcNAc represented by: a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, a pharmacologically acceptable salt of the sugar chain, 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. A method for treating or preventing a disease.

[20] The following formula (2) is applied to an individual in need of improvement or improvement in learning ability:

[Formula 12]

GlcNAcpWManpl-R ¹ (2)

Three

R ³ -Manal ⁷

[Wherein, R ¹ represents a sugar residue, R ² and R ³ are bonded to mannose via a j8 1-2 bond, | 8 1-4 bond or | 8 1-6 bond, May indicate a GlcNAc residue)

A sugar chain having a bisecting GlcNAc represented by: a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, a pharmacologically acceptable salt of the sugar chain, Learning, comprising 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 how to improve or improve Z or memory skills.

[Formula 13]

GlcNAc i-4Manpl-R '(2)

Three

R ³ -Mancd,

A sugar chain having a bisecting GlcNAc represented by: a complex saccharide having the sugar chain in its structure, a derivative of the sugar chain, a derivative of the complex sugar, 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 synthesizing the sugar chain, and a nucleic acid encoding the enzyme.

[22] The following formula (2) for the production of an enhancer or an enhancer of learning and Z or memory ability:

[Wherein, R ¹ represents a sugar residue, and R ² and R ³ are bonded to mannose via a j8 1-2 bond, | 8 1-4 bond or | 8 1-6 bond. Indicates a GlcNAc residue (however, when R ² and R ³ have a GlcNAc residue, one or two R ² and R ³ may be directly bonded to mannose)]