JP5709012B2 - Neuronal differentiation-inducing peptide and use thereof - Google Patents
Neuronal differentiation-inducing peptide and use thereof Download PDFInfo
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- JP5709012B2 JP5709012B2 JP2011508404A JP2011508404A JP5709012B2 JP 5709012 B2 JP5709012 B2 JP 5709012B2 JP 2011508404 A JP2011508404 A JP 2011508404A JP 2011508404 A JP2011508404 A JP 2011508404A JP 5709012 B2 JP5709012 B2 JP 5709012B2
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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Description
本発明は、神経分化誘導性を有するペプチドとその利用に関する。特にそのペプチドを有効成分とする神経分化誘導剤に関する。
なお、本願は２００９年４月１０日に出願された日本国特許出願第２００９−０９５６４０号に基づく優先権を主張しており、当該日本国出願の全内容は本明細書中に参照として援用されている。The present invention relates to a peptide having neuronal differentiation-inducing properties and use thereof. In particular, it relates to a neuronal differentiation inducing agent containing the peptide as an active ingredient.
The present application claims priority based on Japanese Patent Application No. 2009-095640 filed on April 10, 2009, the entire contents of which are incorporated herein by reference. ing.
再生医療分野における一つの課題として神経細胞の再生が挙げられる。例えば、種々の中枢神経系疾患の治療として、神経幹細胞或いは胚性幹細胞（ＥＳ細胞）を利用して神経細胞を再生することが期待されている（特許文献１）。しかし、胚性幹細胞等は入手（採取）が困難である。また、これら幹細胞は患部にそのまま移植されても神経細胞にほとんど分化せず生着も困難である。仮に生着した場合でもグリア細胞に分化してしまうものがほとんどである。
また、神経幹細胞、皮膚幹細胞及び脂肪幹細胞等の体性（成体）幹細胞は、比較的入手が容易な幹細胞であり、これら幹細胞より神経細胞を分化することができれば医療産業上の利用価値は高い。しかし、従来、短時間に且つ高効率にこれら体性幹細胞から神経細胞を分化誘導する方法は未だ確立されておらず、かかる方法の確立、具体的には、そのような目的に適する神経分化誘導剤の開発が望まれている。例えば特許文献２には、ピロリドン誘導体を有効成分とする神経分化誘導剤が記載されているが、体性幹細胞から神経細胞を分化誘導する効果については記載がない。One problem in the field of regenerative medicine is regeneration of nerve cells. For example, as a treatment for various central nervous system diseases, it is expected to regenerate nerve cells using neural stem cells or embryonic stem cells (ES cells) (Patent Document 1). However, it is difficult to obtain (collect) embryonic stem cells and the like. Moreover, even if these stem cells are transplanted as they are to the affected part, they hardly differentiate into nerve cells and are difficult to engraft. Even if engrafted, most of them differentiate into glial cells.
In addition, somatic (adult) stem cells such as neural stem cells, skin stem cells, and adipose stem cells are stem cells that are relatively easily available, and have high utility value in the medical industry if they can be differentiated from these stem cells. However, conventionally, a method for inducing differentiation of nerve cells from these somatic stem cells in a short time and with high efficiency has not yet been established, and establishment of such a method, specifically, induction of neuronal differentiation suitable for such purpose. Development of agents is desired. For example, Patent Document 2 describes a neuronal differentiation inducer containing a pyrrolidone derivative as an active ingredient, but does not describe the effect of inducing differentiation of somatic stem cells.
近年、幹細胞の神経細胞への分化を誘導する機能を有するペプチド（神経分化誘導ペプチド）の利用が注目されている。例えば、特許文献３には、神経幹細胞や皮膚幹細胞からの神経分化を誘導し得るペプチド（ＶＨＬペプチド）が記載されている。 In recent years, the use of peptides having a function of inducing differentiation of stem cells into nerve cells (neural differentiation-inducing peptides) has attracted attention. For example, Patent Document 3 describes a peptide (VHL peptide) that can induce neural differentiation from neural stem cells or skin stem cells.
本発明は、上記特許文献２に記載されるような従前の化学物質を成分とする神経分化誘導剤の開発アプローチとは異なるアプローチにより創出されたものであり、特許文献３に記載されるような既知の神経分化誘導ペプチドよりも高い神経分化誘導活性を有する人工ペプチドの提供を目的とする。また、そのようなペプチドを有効成分とする神経分化誘導剤（薬学的組成物）の提供を他の目的とする。また、そのようなペプチドを使用して神経細胞を生産する方法および神経細胞を発生させる方法を提供することを他の目的とする。 The present invention was created by an approach different from the development approach of a neuronal differentiation inducer containing a chemical substance as a component as described in Patent Document 2, and is described in Patent Document 3. An object is to provide an artificial peptide having higher neuronal differentiation-inducing activity than known neuronal differentiation-inducing peptides. Another object is to provide a neuronal differentiation inducer (pharmaceutical composition) containing such a peptide as an active ingredient. Another object of the present invention is to provide a method for producing nerve cells using such a peptide and a method for generating nerve cells.
本発明によって提供される神経分化誘導ペプチドは、それ単独で自然界において神経分化誘導ペプチドとして存在するものではない人為的に設計された合成ペプチドである。
本発明者は、低酸素誘導因子（ＨＩＦ−１：Hypoxia-Inducible Factor 1）を構成する二つのサブユニット（即ちＨＩＦ−１αとＨＩＦ−１β）のうちの一つであるＨＩＦ−１αのＰＡＳ−Ａドメイン（即ちPer-Arnt-Sim ホモロジードメインのサブドメインＡ）に結合するＨＩＦ−１α結合タンパク質であって活性化プロテインＣキナーゼ受容体として同定されたタンパク質「ＲＡＣＫ１(a Receptor of Activated Protein Kinase C)」に着目した。さらに、かかるＲＡＣＫ１タンパク質中のＷＤ６ドメイン、即ちエロンジンＣ(Elongin C)に結合し得る領域（アミノ酸配列）に着目した（非特許文献１〜３参照)。The neuronal differentiation-inducing peptide provided by the present invention is an artificially designed synthetic peptide that alone does not exist as a neuronal differentiation-inducing peptide in nature.
The present inventor has developed a PAS- of HIF-1α that is one of two subunits (ie, HIF-1α and HIF-1β) that constitutes hypoxia-inducible factor 1 (HIF-1). A protein “RACK1 (a Receptor of Activated Protein Kinase C) that is an HIF-1α binding protein that binds to the A domain (ie, subdomain A of the Per-Arnt-Sim homology domain) and has been identified as an activated protein C kinase receptor ”. Furthermore, the WD6 domain in the RACK1 protein, that is, a region (amino acid sequence) capable of binding to Elongin C was focused (see Non-Patent Documents 1 to 3).
そして、種々の哺乳動物細胞や鳥類、両生類等の細胞に存在するＲＡＣＫ１の上記ＷＤ６ドメイン中の部分アミノ酸配列を含むようにして作製した合成ペプチドが、種々の幹細胞に対して高い神経分化誘導活性を有することを見出した。
さらに、本発明者は、核小体局在シグナル（ＮｏＬＳ：Nucleolar localization signal）として知られるアミノ酸配列（非特許文献４参照）が細胞外から核内（典型的には核小体）へのペプチド移行に関与するアミノ酸配列であることを見出し、該アミノ酸配列を含むように構成された合成ペプチドを使用することにより、幹細胞から神経細胞への分化効率を著しく高め得ることを見出して本発明を完成するに至った。A synthetic peptide prepared by including a partial amino acid sequence in the WD6 domain of RACK1 present in various mammalian cells, birds, amphibians and the like has high neuronal differentiation-inducing activity against various stem cells. I found.
Furthermore, the present inventor has found that an amino acid sequence known as a nucleolus localization signal (NoLS: Nucleolar localization signal) (see Non-Patent Document 4) is a peptide from outside the nucleus to the nucleus (typically a nucleolus). The present invention was completed by finding that the amino acid sequence involved in the transition and using a synthetic peptide configured to contain the amino acid sequence can significantly increase the efficiency of differentiation from stem cells to neurons. It came to do.
ここで開示される神経分化誘導ペプチドは、少なくとも一種の幹細胞を神経細胞に分化誘導し得るペプチド（以下「神経分化誘導ペプチド」ともいう。）である。
即ち、本発明は、そのようなペプチドとして、ＲＡＣＫ１に含まれる部分配列であるＲＡＣＫ１関連アミノ配配列として以下のアミノ酸配列：
Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-（Ｉ又はＶ）-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ
を含む人為的に合成されたペプチドを提供する。
なお、本明細書中に記載されるアミノ酸配列中のハイフン（−）は隣接する二つのアミノ酸残基間のペプチド結合を示している（以下同じ。）。また、本明細書中に記載されるアミノ酸配列は、常に左側がＮ末端側であり右側がＣ末端側である。The neuronal differentiation-inducing peptide disclosed herein is a peptide that can induce differentiation of at least one kind of stem cell into a neuron (hereinafter also referred to as “neural differentiation-inducing peptide”).
That is, the present invention provides the following amino acid sequence as a RACK1-related amino acid sequence, which is a partial sequence contained in RACK1, as such a peptide:
T-L-D-G-G-D- (I or V) -I-N-A-L-C-F-S
An artificially synthesized peptide is provided.
In addition, the hyphen (-) in the amino acid sequence described in the present specification indicates a peptide bond between two adjacent amino acid residues (the same applies hereinafter). In the amino acid sequences described herein, the left side is always the N-terminal side and the right side is the C-terminal side.
ここで開示されるペプチドの好ましい一態様は、上記ＲＡＣＫ１関連アミノ酸配列に加えて、核小体局在シグナル（ＮｏＬＳ）を構成する以下のアミノ酸配列：
Ｋ-Ｋ-Ｒ-Ｔ-Ｌ-Ｒ-Ｋ-Ｎ-Ｄ-Ｒ-Ｋ-Ｋ-Ｒ（配列番号１）
をさらに含む人為的に合成されたペプチドである。
好ましくは、上記合成ペプチドを構成する全アミノ酸残基数は５０以下である。In addition to the RACK1-related amino acid sequence described above, a preferred embodiment of the peptide disclosed herein includes the following amino acid sequence constituting a nucleolar localization signal (NoLS):
KK-R-T-L-R-K-N-D-R-K-K-R (SEQ ID NO: 1)
Is an artificially synthesized peptide.
Preferably, the total number of amino acid residues constituting the synthetic peptide is 50 or less.
また、ここで開示されるペプチドの好ましい一態様は、上記ＲＡＣＫ１関連アミノ酸配列として以下のアミノ酸配列：
Ｌ-Ｙ-Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-（Ｉ又はＶ）-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ-Ｐ-Ｎ-Ｒ-Ｙ-Ｗ-Ｌ
を含む。One preferred embodiment of the peptide disclosed herein is the following amino acid sequence as the RACK1-related amino acid sequence:
L-Y-T-L-D-G-G-D- (I or V) -I-N-A-L-C-F-S-P-N-R-Y-W-L
including.
また、ここで開示される神経分化誘導ペプチドの特に好ましい一態様は、上記核小体局在シグナル（ＮｏＬＳ）を構成するアミノ酸配列のＮ末端側またはＣ末端側に隣接して上記ＲＡＣＫ１関連アミノ酸配列を含む。
具体的には、上記ＲＡＣＫ１関連アミノ酸配列として、
Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-Ｉ-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ（配列番号２）、
Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-Ｖ-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ（配列番号３）、
Ｌ-Ｙ-Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-Ｉ-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ-Ｐ-Ｎ-Ｒ-Ｙ-Ｗ-Ｌ（配列番号４）、および、
Ｌ-Ｙ-Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-Ｖ-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ-Ｐ-Ｎ-Ｒ-Ｙ-Ｗ-Ｌ（配列番号５）が挙げられる。なお、これらＲＡＣＫ１関連アミノ酸配列は、配列番号２〜５の各々に記載されたとおりのアミノ酸配列の他、これらアミノ酸配列に部分的な改変が施された改変アミノ酸配列を包含する。
本発明によって提供される神経分化誘導ペプチドの好ましい具体例として、配列番号６、配列番号７、配列番号８および配列番号９のうちのいずれかに示すアミノ酸配列から成る合成ペプチドが挙げられる。In addition, a particularly preferred embodiment of the neuronal differentiation-inducing peptide disclosed herein is the RACK1-related amino acid sequence adjacent to the N-terminal side or C-terminal side of the amino acid sequence constituting the nucleolar localization signal (NoLS). including.
Specifically, as the RACK1-related amino acid sequence,
T-L-D-G-G-D-I-I-N-A-L-C-F-S (SEQ ID NO: 2),
T-L-D-G-G-D-V-I-N-A-L-C-F-S (SEQ ID NO: 3),
L-Y-T-L-D-G-G-D-I-I-N-A-L-C-F-S-P-N-R-Y-W-L (SEQ ID NO: 4), and ,
LY-T-L-D-G-G-D-V-I-N-A-L-C-F-S-P-N-R-Y-W-L (SEQ ID NO: 5) It is done. These RACK1-related amino acid sequences include amino acid sequences as described in SEQ ID NOs: 2 to 5, and modified amino acid sequences in which these amino acid sequences are partially modified.
Preferable specific examples of the neuronal differentiation-inducing peptide provided by the present invention include a synthetic peptide consisting of the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
また、本発明は、他の側面として、ここで開示されるいずれかの神経分化誘導ペプチドと、薬学上許容され得る少なくとも一種の担体とを含有する、少なくとも一種の幹細胞を神経細胞に分化誘導し得る神経分化誘導剤を提供する。
また、本発明は他の側面として、ここで開示される神経分化誘導剤若しくは神経分化誘導ペプチドを利用する種々の方法を提供する。
即ち、本発明は、少なくとも一種の細胞材料から神経細胞を生産する方法を提供する。この方法は、ここで開示されるいずれかの合成ペプチド若しくは該合成ペプチドを含む神経分化誘導剤（薬学的組成物）を用意し、該ペプチド若しくは該神経分化誘導剤を上記細胞材料に供給することを特徴とする。
また、本発明は、生体又は生組織中に神経細胞を発生させる方法を提供する。この方法は、ここで開示されるいずれかの合成ペプチド若しくは該合成ペプチドを含む神経分化誘導剤（薬学的組成物）を用意し、生体または生体外に一時的又は永久的に摘出された生組織に上記合成ペプチド若しくは該合成ペプチドを含む神経分化誘導剤（薬学的組成物）を付与することを特徴とする。Further, the present invention, as another aspect, induces differentiation of at least one stem cell containing any one of the neuronal differentiation-inducing peptides disclosed herein and at least one pharmaceutically acceptable carrier into a nerve cell. The obtained neuronal differentiation inducer is provided.
As another aspect, the present invention provides various methods using the neuronal differentiation inducer or neuronal differentiation-inducing peptide disclosed herein.
That is, the present invention provides a method for producing nerve cells from at least one cell material. In this method, any of the synthetic peptides disclosed herein or a neuronal differentiation inducer (pharmaceutical composition) containing the synthetic peptide is prepared, and the peptide or the neuronal differentiation inducer is supplied to the cell material. It is characterized by.
The present invention also provides a method for generating nerve cells in a living body or living tissue. In this method, any of the synthetic peptides disclosed herein or a neuronal differentiation inducer (pharmaceutical composition) containing the synthetic peptide is prepared, and a living tissue temporarily or permanently removed from a living body or in vitro. Or a neuronal differentiation inducer (pharmaceutical composition) containing the synthetic peptide.
また、本発明は、ここで開示されるいずれかの合成ペプチドをコードするヌクレオチド配列及び／又は該配列と相補的なヌクレオチド配列を含む、天然に存在しない人為的に設計されたポリヌクレオチド（例えばそれら配列により実質的に構成されるポリヌクレオチド）を提供する。
好ましいポリヌクレオチドとして、配列番号６〜９のうちのいずれかに示されるアミノ酸配列をコードするヌクレオチド配列及び／又は該配列と相補的なヌクレオチド配列を含むポリヌクレオチド（例えばそれら配列により実質的に構成されるポリヌクレオチド）が挙げられる。The present invention also includes non-naturally occurring artificially designed polynucleotides comprising nucleotide sequences encoding any of the synthetic peptides disclosed herein and / or nucleotide sequences complementary thereto (eg, those A polynucleotide substantially constituted by the sequence).
As a preferred polynucleotide, a polynucleotide comprising the nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NOs: 6 to 9 and / or a nucleotide sequence complementary to the sequence (for example, substantially constituted by these sequences) Polynucleotide).
本発明の神経分化誘導ペプチドは、上述のとおり、ＲＡＣＫ１関連アミノ酸配列（好ましくはＲＡＣＫ１関連アミノ酸配列に加えて核小体局在シグナル（ＮｏＬＳ）を構成するアミノ酸配列）を含む合成ペプチドであるため容易に製造することができる。このため、所望する量のペプチド（延いては神経分化誘導剤）を容易に調製することができる。
また、本発明によると、そのような神経分化誘導ペプチド（神経分化誘導剤）を利用することにより、従来は困難であった非神経細胞（典型的には脂肪幹細胞、皮膚幹細胞等の体性幹細胞、或いは胚性幹細胞）を神経細胞に分化誘導することが容易に実現される。このため、比較的大量に調達し得る細胞材料（脂肪幹細胞等）を使用して、用途（例えば、神経の再生が要求されるような神経疾患の治療）に応じて所望される量の神経細胞を供給することができる。或いはまた、神経再生が必要な患部や体外に摘出した一時的又は永久的な生組織（細胞塊等の培養物を含む）に適当量を投与することにより、神経細胞の発生を実現することができる。As described above, the neuronal differentiation-inducing peptide of the present invention is easy because it is a synthetic peptide containing a RACK1-related amino acid sequence (preferably an amino acid sequence constituting a nucleolar localization signal (NoLS) in addition to the RACK1-related amino acid sequence). Can be manufactured. Therefore, a desired amount of peptide (and thus a neuronal differentiation inducer) can be easily prepared.
In addition, according to the present invention, non-neuronal cells (typically somatic stem cells such as adipose stem cells and skin stem cells) that have been difficult in the past by using such a neuronal differentiation-inducing peptide (neurogenesis differentiation-inducing agent). Or embryonic stem cells) can be easily induced to differentiate into neurons. For this reason, using a cell material (such as adipose stem cell) that can be procured in a relatively large amount, a desired amount of nerve cells depending on the application (for example, treatment of a nerve disease requiring nerve regeneration) Can be supplied. Alternatively, the generation of nerve cells can be realized by administering an appropriate amount to an affected part that needs nerve regeneration or a temporary or permanent living tissue (including a culture such as a cell mass) extracted outside the body. it can.
以下、本発明の好適な実施形態を説明する。なお、本明細書において特に言及している事項（例えば神経分化誘導ペプチドの一次構造や鎖長）以外の事柄であって本発明の実施に必要な事柄（例えばペプチド合成、ポリヌクレオチド合成、ペプチドを成分とする神経分化誘導剤（薬剤組成物）の調製に関するような一般的事項）は、医学、薬学、有機化学、生化学、遺伝子工学、タンパク質工学、分子生物学、衛生学等の分野における従来技術に基づく当業者の設計事項として把握され得る。本発明は、本明細書に開示されている内容と当該分野における技術常識とに基づいて実施することができる。なお、以下の説明では、場合に応じてアミノ酸をIUPAC-IUBガイドラインで示されたアミノ酸に関する命名法に準拠した１文字表記（但し配列表では３文字表記）で表す。
また、本明細書中で引用されている全ての文献の全ての内容は本明細書中に参照として組み入れられている。Hereinafter, preferred embodiments of the present invention will be described. It should be noted that matters other than matters specifically mentioned in the present specification (for example, the primary structure and chain length of neuronal differentiation-inducing peptides) and matters necessary for carrying out the present invention (for example, peptide synthesis, polynucleotide synthesis, peptide General matters such as preparation of nerve differentiation-inducing agents (pharmaceutical compositions) as ingredients) are conventional in the fields of medicine, pharmacy, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, hygiene, etc. It can be grasped as a design matter of those skilled in the art based on the technology. The present invention can be carried out based on the contents disclosed in this specification and common technical knowledge in the field. In the following description, amino acids are represented by one-letter code (in the sequence table, three-letter code) based on the nomenclature related to amino acids shown in the IUPAC-IUB guidelines.
In addition, the entire contents of all documents cited in this specification are incorporated herein by reference.
本明細書において「人為的に合成された神経分化誘導ペプチド」とは、そのペプチド鎖がそれのみで独立して自然界に安定的に存在するものではなく、人為的な化学合成或いは生合成（即ち遺伝子工学に基づく生産）によって製造され、所定の系（例えば神経分化誘導剤を構成する組成物）の中で安定して存在し得るペプチド断片をいう。
本明細書において「ペプチド」とは、複数のペプチド結合を有するアミノ酸ポリマーを指す用語であり、ペプチド鎖に含まれるアミノ酸残基の数によって限定されないが、典型的には全アミノ酸残基数が１００以下、好ましくは５０以下である。In the present specification, the “artificially synthesized neuronal differentiation-inducing peptide” does not mean that the peptide chain exists independently and stably in nature, but artificial chemical synthesis or biosynthesis (ie, A peptide fragment that is produced by genetic engineering) and can exist stably in a predetermined system (for example, a composition constituting a neuronal differentiation inducer).
As used herein, “peptide” is a term that refers to an amino acid polymer having a plurality of peptide bonds, and is not limited by the number of amino acid residues contained in the peptide chain. Hereinafter, it is preferably 50 or less.
本明細書において「アミノ酸残基」とは、特に言及する場合を除いて、ペプチド鎖のＮ末端アミノ酸及びＣ末端アミノ酸を包含する用語である。また、本明細書において所定のアミノ酸配列に対して「部分的な改変が施されたアミノ酸配列（改変アミノ酸配列）」とは、当該所定のアミノ酸配列が有する神経分化誘導性を損なうことなく、１個または数個（例えば２，３個）のアミノ酸残基が置換、欠失及び／又は付加（挿入）されて形成されたアミノ酸配列をいう。例えば、１個又は数個（典型的には２個又は３個）のアミノ酸残基が保守的に置換したいわゆる同類置換（conservative amino acid replacement）によって生じた配列（例えば塩基性アミノ酸残基が別の塩基性アミノ酸残基に置換した配列）、或いは、所定のアミノ酸配列について１個又は数個（典型的には２個又は３個）のアミノ酸残基が付加（挿入）した若しくは欠失した配列等は、本明細書でいう「部分的な改変が施された配列（改変アミノ酸配列）」に包含される典型例である。
また、本明細書において「ポリヌクレオチド」とは、複数のヌクレオチドがリン酸ジエステル結合で結ばれたポリマー（核酸）を指す用語であり、ヌクレオチドの数によって限定されない。種々の長さのＤＮＡフラグメント及びＲＮＡフラグメントが本明細書におけるポリヌクレオチドに包含される。また、「人為的に設計されたポリヌクレオチド」とは、そのヌクレオチド鎖（全長）がそれ単独で自然界に存在するものではなく、化学合成或いは生合成（即ち遺伝子工学に基づく生産）によって人為的に合成されたポリヌクレオチドをいう。In the present specification, “amino acid residue” is a term including the N-terminal amino acid and the C-terminal amino acid of a peptide chain, unless otherwise specified. Further, in this specification, a “partially modified amino acid sequence (modified amino acid sequence)” with respect to a predetermined amino acid sequence refers to 1 1 without impairing the neuronal differentiation-inducing property of the predetermined amino acid sequence. An amino acid sequence formed by substitution, deletion and / or addition (insertion) of one or several (for example, two or three) amino acid residues. For example, sequences generated by so-called conservative amino acid replacement (for example, basic amino acid residues are separated) in which one or several (typically 2 or 3) amino acid residues are conservatively substituted. Or a sequence in which one or several (typically 2 or 3) amino acid residues have been added (inserted) or deleted with respect to a predetermined amino acid sequence. Are typical examples included in the “partially modified sequence (modified amino acid sequence)” as used herein.
In the present specification, the “polynucleotide” is a term indicating a polymer (nucleic acid) in which a plurality of nucleotides are linked by phosphodiester bonds, and is not limited by the number of nucleotides. Various lengths of DNA and RNA fragments are encompassed by the polynucleotides herein. In addition, “artificially designed polynucleotide” means that the nucleotide chain (full length) does not exist in nature alone, but is artificially generated by chemical synthesis or biosynthesis (ie, production based on genetic engineering). It refers to a synthesized polynucleotide.
本発明者は、ヒト、マウス等の哺乳動物、ニワトリ等の鳥類、その他カエル等の脊椎動物由来のＲＡＣＫ１に含まれる部分アミノ酸配列、詳しくは、エロンジンＣとの結合に関与する領域（主としてＷＤ６ドメイン）中の一部のアミノ酸配列（ＲＡＣＫ１関連アミノ酸配列）を利用して合成した比較的短いペプチドが神経分化誘導活性を発揮し得ることを見出した。そのＲＡＣＫ１関連アミノ酸配列の典型例を配列番号２〜５に示す。
具体的には、配列番号２は、ヒトを含む哺乳動物（ヒト、マウス、ラット、豚、等）由来のＲＡＣＫ１のＮ末端より第２２９番目のアミノ酸残基から第２４２番目のアミノ酸残基までの合計１４アミノ酸残基から成るアミノ酸配列である。また、配列番号３は、カエル由来のＲＡＣＫ１のＮ末端より第２２９番目のアミノ酸残基から第２４２番目のアミノ酸残基までの合計１４アミノ酸残基から成るアミノ酸配列である。配列番号２に示すアミノ酸配列と配列番号３に示すアミノ酸配列とは、Ｎ末端から第２３５番目のアミノ酸残基がイソロイシン（配列番号２）からバリン（配列番号３）に置換されていることを除き、共通である。
また、配列番号４は、配列番号２のアミノ酸配列を全て包含するアミノ酸配列であり、ヒトを含む哺乳動物（ヒト、マウス、ラット、豚、等）由来のＲＡＣＫ１のＮ末端より第２２７番目のアミノ酸残基から第２４８番目のアミノ酸残基までの合計２２アミノ酸残基から成るアミノ酸配列である。また、配列番号５は、配列番号３のアミノ酸配列を全て包含するアミノ酸配列であり、カエル由来のＲＡＣＫ１のＮ末端より第２２７番目のアミノ酸残基から第２４８番目のアミノ酸残基までの合計２２アミノ酸残基から成るアミノ酸配列である。配列番号４に示すアミノ酸配列と配列番号５に示すアミノ酸配列とは、Ｎ末端より第２３５番目のアミノ酸残基がイソロイシン（配列番号２）からバリン（配列番号３）に置換されていることを除き、共通である。The present inventor has developed a partial amino acid sequence contained in RACK1 derived from mammals such as humans and mice, birds such as chickens, and vertebrates such as frogs, specifically, a region involved in binding to elongin C (mainly the WD6 domain). It was found that a relatively short peptide synthesized using a part of the amino acid sequence (RACK1-related amino acid sequence) in FIG. Typical examples of the RACK1-related amino acid sequences are shown in SEQ ID NOs: 2 to 5.
Specifically, SEQ ID NO: 2 is from the 229th amino acid residue to the 242nd amino acid residue from the N-terminus of RACK1 derived from mammals including humans (human, mouse, rat, pig, etc.). It is an amino acid sequence consisting of a total of 14 amino acid residues. SEQ ID NO: 3 is an amino acid sequence consisting of a total of 14 amino acid residues from the 229th amino acid residue to the 242nd amino acid residue from the N-terminus of frog-derived RACK1. The amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 3 except that the 235th amino acid residue from the N-terminus is replaced by isoleucine (SEQ ID NO: 2) to valine (SEQ ID NO: 3). , Is common.
SEQ ID NO: 4 is an amino acid sequence including all amino acid sequences of SEQ ID NO: 2, and the 227th amino acid from the N-terminus of RACK1 derived from mammals including humans (human, mouse, rat, pig, etc.) It is an amino acid sequence consisting of a total of 22 amino acid residues from the residue to the 248th amino acid residue. SEQ ID NO: 5 is an amino acid sequence encompassing all amino acid sequences of SEQ ID NO: 3, and a total of 22 amino acids from the 227th amino acid residue to the 248th amino acid residue from the N-terminus of frog-derived RACK1 An amino acid sequence consisting of residues. The amino acid sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 5, except that the 235th amino acid residue from the N-terminus is replaced with isoline (SEQ ID NO: 2) to valine (SEQ ID NO: 3). , Is common.
上記配列番号２及び配列番号３に示すＲＡＣＫ１関連アミノ酸配列と、配列番号４及び配列番号５に示すＲＡＣＫ１関連アミノ酸配列の関係（対比）から、以下の（１）〜（３）に示すアミノ酸配列もまた本発明に関するＲＡＣＫ１関連アミノ酸配列として好ましい例であることは、当業者には容易に理解される。即ち、
（１）．配列番号２または配列番号３に示すアミノ酸配列のＮ末端側に、アミノ酸残基「Ｙ」または「Ｌ-Ｙ」が付加されたアミノ酸配列；
（２）．配列番号２または配列番号３に示すアミノ酸配列のＣ末端側に、アミノ酸残基「Ｐ-Ｎ-Ｒ-Ｙ-Ｗ-Ｌ」または「Ｐ-Ｎ-Ｒ-Ｙ-Ｗ」または「Ｐ-Ｎ-Ｒ-Ｙ」または「Ｐ-Ｎ-Ｒ」または「Ｐ-Ｎ」または「Ｐ」が付加されたアミノ酸配列；
（３）．配列番号２または配列番号３に示すアミノ酸配列のＮ末端側にはアミノ酸残基「Ｙ」または「Ｌ-Ｙ」が付加され、且つ、Ｃ末端側にはアミノ酸残基「Ｐ-Ｎ-Ｒ-Ｙ-Ｗ-Ｌ」または「Ｐ-Ｎ-Ｒ-Ｙ-Ｗ」または「Ｐ-Ｎ-Ｒ-Ｙ」または「Ｐ-Ｎ-Ｒ」または「Ｐ-Ｎ」または「Ｐ」が付加されたアミノ酸配列。From the relationship (contrast) between the RACK1-related amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 and the RACK1-related amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5, the amino acid sequences shown in the following (1) to (3) are also Moreover, it is easily understood by those skilled in the art that this is a preferable example of the RACK1-related amino acid sequence according to the present invention. That is,
(1). An amino acid sequence in which an amino acid residue “Y” or “LY” is added to the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3;
(2). In the C-terminal side of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, the amino acid residues “PNNRYWL” or “PNNRYW” or “PN” -R—Y ”or“ PN—R ”or“ PN ”or“ P ”added amino acid sequence;
(3). The amino acid residue “Y” or “LY” is added to the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, and the amino acid residue “P—N—R— is added to the C-terminal side. Y-W-L "or" PN-R-Y-W "or" PN-R-Y "or" PN-R "or" PN "or" P "is added Amino acid sequence.
設計される神経分化誘導ペプチドは、前記ＲＡＣＫ１関連アミノ酸配列またはその改変アミノ酸配列のみから成るペプチドであってもよいが、神経分化誘導活性の向上の観点からは、いわゆる細胞膜通過ドメイン（タンパク質導入ドメイン：Protein Transduction Domain）を構成するアミノ酸配列の利用が好ましい。好適例を配列番号１０，１１及び１２に示す。配列番号１０は、ＨＩＶのＴＡＴに含まれるタンパク質導入ドメインのアミノ酸配列と該配列から成るペプチドを示している。配列番号１１は、前記ＴＡＴを改変したタンパク質導入ドメイン（ＰＴＤ４）のアミノ酸配列と該配列から成るペプチドを示している。配列番号１２は、ショウジョウバエ（Drosophila）の変異体AntennapediaのＡＮＴの関連アミノ酸配列と該配列から成るペプチドを示している。なお、配列表に示した上述の細胞膜通過ドメインはあくまでも例示であり、使用可能なドメインはこれらに限定されない。本発明の実施に使用可能な様々な細胞膜通過ドメインが本願出願当時に出版されている数々の文献に記載されている。それら細胞膜通過ドメインのアミノ酸配列は一般的な検索手段によって容易に知ることができる。
特に、以下のアミノ酸配列：
Ｋ-Ｋ-Ｒ-Ｔ-Ｌ-Ｒ-Ｋ-Ｎ-Ｄ-Ｒ-Ｋ-Ｋ-Ｒ（配列番号１）
の利用が好ましい。
本発明者は、上記非特許文献４に記載されるように核小体局在シグナル（ＮｏＬＳ）として知られる上記配列番号１に示すアミノ酸配列と、目的とする他のアミノ酸配列（ペプチドモチーフ）を構成するアミノ酸配列とを含むペプチドを合成し、培養中の真核細胞に添加したところ、当該ペプチドが高効率に対象細胞の細胞膜を通過し得ること、さらには高効率に核膜を通過し得ることを見出した。
即ち、本発明によると、目的とするＲＡＣＫ１関連アミノ酸配列を上記配列番号１に示すアミノ酸配列（以下「核小体局在シグナル関連アミノ酸配列」ともいう。）と組み合わせて得られた人工ペプチドを構築（合成) し、対象とする真核細胞に添加することによって、当該人工ペプチドを真核細胞の外部（細胞膜の外側）から核内（好ましくは核小体）に高効率に移送することができる。The designed neuronal differentiation-inducing peptide may be a peptide consisting only of the RACK1-related amino acid sequence or its modified amino acid sequence, but from the viewpoint of improving the neuronal differentiation-inducing activity, a so-called cell membrane translocation domain (protein transduction domain: Use of an amino acid sequence constituting a protein transduction domain is preferred. Preferred examples are shown in SEQ ID NOs: 10, 11 and 12. SEQ ID NO: 10 shows the amino acid sequence of the protein transduction domain contained in HIV TAT and a peptide comprising the sequence. SEQ ID NO: 11 shows the amino acid sequence of the protein transduction domain (PTD4) obtained by modifying the TAT, and a peptide comprising the sequence. SEQ ID NO: 12 shows the related amino acid sequence of ANT of Drosophila mutant Antennapedia and a peptide comprising the sequence. In addition, the above-mentioned cell membrane passage domain shown to the sequence table is an illustration to the last, and the domain which can be used is not limited to these. Various cell membrane translocation domains that can be used in the practice of the present invention are described in numerous publications published at the time of filing this application. The amino acid sequences of these cell membrane translocation domains can be easily known by general search means.
In particular, the following amino acid sequence:
KK-R-T-L-R-K-N-D-R-K-K-R (SEQ ID NO: 1)
Is preferably used.
As described in Non-Patent Document 4 above, the present inventor has obtained the amino acid sequence shown in SEQ ID NO: 1 known as a nucleolus localization signal (NoLS) and other desired amino acid sequence (peptide motif). When a peptide containing a constituent amino acid sequence is synthesized and added to a eukaryotic cell in culture, the peptide can pass through the cell membrane of the target cell with high efficiency, and further can pass through the nuclear membrane with high efficiency. I found out.
That is, according to the present invention, an artificial peptide obtained by combining the target RACK1-related amino acid sequence with the amino acid sequence shown in SEQ ID NO: 1 (hereinafter also referred to as “nuclear body localization signal-related amino acid sequence”) is constructed. (Synthesis) and by adding to the target eukaryotic cell, the artificial peptide can be efficiently transferred from the outside of the eukaryotic cell (outside the cell membrane) to the inside of the nucleus (preferably the nucleolus). .
本発明によって提供される神経分化誘導ペプチドは、少なくとも一つのアミノ酸残基がアミド化されているものが好ましい。アミノ酸残基（典型的にはペプチド鎖のＣ末端アミノ酸残基）のカルボキシル基のアミド化により、神経分化誘導ペプチドの構造安定性（例えばプロテアーゼ耐性）を向上させ得る。
神経分化誘導ペプチドは、ペプチド鎖を構成する全アミノ酸残基数が１００以下が望ましく、５０以下が好ましい。このような鎖長の短いペプチドは、化学合成が容易であり、容易に神経分化誘導ペプチドを提供することができる。なお、ペプチドのコンホメーション（立体構造）については、使用する環境下で神経分化誘導性を発揮する限りにおいて、特に限定されるものではないが、免疫原（抗原）になり難いという観点から直鎖状又はへリックス状のものが好ましい。このような形状のペプチドはエピトープを構成し難い。かかる観点から、神経分化誘導剤に適用する神経分化誘導ペプチドとしては、直鎖状であり比較的低分子量（典型的には５０以下（特に４０以下）のアミノ酸残基数）のものが好適である。The neuronal differentiation-inducing peptide provided by the present invention is preferably one in which at least one amino acid residue is amidated. By amidating the carboxyl group of an amino acid residue (typically the C-terminal amino acid residue of the peptide chain), the structural stability (eg, protease resistance) of the neuronal differentiation-inducing peptide can be improved.
In the neuronal differentiation-inducing peptide, the total number of amino acid residues constituting the peptide chain is desirably 100 or less, and preferably 50 or less. Such a peptide having a short chain length is easy to chemically synthesize, and can easily provide a neuronal differentiation-inducing peptide. The peptide conformation (three-dimensional structure) is not particularly limited as long as it exhibits neuronal differentiation inducibility in the environment in which it is used, but it is straightforward from the viewpoint that it is difficult to become an immunogen (antigen). A chain or helix is preferred. Such a peptide is unlikely to constitute an epitope. From this point of view, the neuronal differentiation-inducing peptide applied to the neuronal differentiation-inducing agent is preferably a linear and relatively low molecular weight (typically 50 or less (particularly 40 or less) amino acid residues). is there.
全体のアミノ酸配列に対するＲＡＣＫ１関連アミノ酸配列の占める割合（即ちペプチド鎖を構成する全アミノ酸残基数に占めるＲＡＣＫ１関連アミノ酸配列部分を構成するアミノ酸残基数の個数％）は、神経分化誘導活性を失わない限り特に限定されないが、当該割合は４０％以上が望ましく、５０％以上が好ましい。なお、本発明の神経分化誘導ペプチドとしては、全てのアミノ酸残基がＬ型アミノ酸であるものが好ましいが、神経分化誘導活性を失わない限りにおいて、アミノ酸残基の一部又は全部がＤ型アミノ酸に置換されているものであってもよい。 The ratio of the RACK1-related amino acid sequence to the total amino acid sequence (that is, the number of amino acid residues constituting the RACK1-related amino acid sequence in the total number of amino acid residues constituting the peptide chain) loses the neuronal differentiation-inducing activity. Although there is no particular limitation as long as there is no limit, the proportion is desirably 40% or more, and preferably 50% or more. The neuronal differentiation-inducing peptide of the present invention is preferably one in which all amino acid residues are L-type amino acids. However, as long as the neuronal differentiation-inducing activity is not lost, part or all of the amino acid residues are D-type amino acids. May be substituted.
本発明の神経分化誘導ペプチドは、神経分化誘導性を失わない限りにおいて、ＲＡＣＫ１関連アミノ酸配列ならびに核小体局在シグナル関連アミノ酸配列に含まれ得ない配列を部分的に含み得る。特に限定するものではないが、かかる部分配列としてはペプチド鎖におけるＲＡＣＫ１関連アミノ酸配列部分の３次元形状（典型的には直鎖形状）を維持し得る配列が好ましい。 The neuronal differentiation-inducing peptide of the present invention may partially contain a RACK1-related amino acid sequence as well as a sequence that cannot be included in the nucleolus localization signal-related amino acid sequence as long as the neuronal differentiation-inducing property is not lost. Although not particularly limited, such a partial sequence is preferably a sequence capable of maintaining the three-dimensional shape (typically linear shape) of the RACK1-related amino acid sequence portion in the peptide chain.
ここで開示される神経分化誘導ペプチドのうちペプチド鎖の比較的短いものは、一般的な化学合成法に準じて容易に製造することができる。例えば、従来公知の固相合成法又は液相合成法のいずれを採用してもよい。アミノ基の保護基としてＢｏｃ（t-butyloxycarbonyl）或いはＦｍｏｃ（9-fluorenylmethoxycarbonyl）を適用した固相合成法が好適である。
ここで開示される神経分化誘導ペプチドは、市販のペプチド合成機（例えば、Intavis AG社、Applied Biosystems社等から入手可能である。）を用いた固相合成法により、所望するアミノ酸配列、修飾（Ｃ末端アミド化等）部分を有するペプチド鎖を合成することができる。Among the neuronal differentiation-inducing peptides disclosed herein, a peptide having a relatively short peptide chain can be easily produced according to a general chemical synthesis method. For example, any conventionally known solid phase synthesis method or liquid phase synthesis method may be employed. A solid phase synthesis method in which Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) is applied as an amino-protecting group is preferred.
The neuronal differentiation-inducing peptide disclosed here can be obtained by a solid-phase synthesis method using a commercially available peptide synthesizer (for example, available from Intavis AG, Applied Biosystems, etc.). A peptide chain having a moiety such as a C-terminal amidation can be synthesized.
或いは、遺伝子工学的手法に基づいて神経分化誘導ペプチドを生合成してもよい。このアプローチは、ペプチド鎖の比較的長いポリペプチドを製造する場合に好適である。すなわち、所望する神経分化誘導ペプチドのアミノ酸配列をコードするヌクレオチド配列（ＡＴＧ開始コドンを含む。）のＤＮＡを合成する。そして、このＤＮＡと該アミノ酸配列を宿主細胞内で発現させるための種々の調節エレメント（プロモーター、リボゾーム結合部位、ターミネーター、エンハンサー、発現レベルを制御する種々のシスエレメントを包含する。）とから成る発現用遺伝子構築物を有する組換えベクターを、宿主細胞に応じて構築する。
一般的な技法によって、この組換えベクターを所定の宿主細胞（例えばイースト、昆虫細胞、植物細胞、動物（哺乳類）細胞）に導入し、所定の条件で当該宿主細胞又は該細胞を含む組織や個体を培養する。このことにより、目的とするポリペプチドを細胞内で発現、生産させることができる。そして、宿主細胞（分泌された場合は培地中）からポリペプチドを単離し、精製することによって、目的の神経分化誘導ペプチドを得ることができる。一般的な技法によって、この組換えベクターを所定の宿主細胞（例えばイースト、昆虫細胞、植物細胞、哺乳類細胞）に導入し、所定の条件で当該宿主細胞又は該細胞を含む組織や個体を培養する。このことにより、目的とするポリペプチドを細胞内で発現、生産させることができる。そして、宿主細胞（分泌された場合は培地中）からポリペプチドを単離し、精製することによって、目的の神経分化誘導ペプチドを得ることができる。
なお、組換えベクターの構築方法及び構築した組換えベクターの宿主細胞への導入方法等は、当該分野で従来から行われている方法をそのまま採用すればよく、かかる方法自体は特に本発明を特徴付けるものではないため、詳細な説明は省略する。Alternatively, neuronal differentiation-inducing peptides may be biosynthesized based on genetic engineering techniques. This approach is preferred when producing polypeptides with relatively long peptide chains. That is, DNA having a nucleotide sequence (including the ATG start codon) encoding the amino acid sequence of a desired neuronal differentiation-inducing peptide is synthesized. An expression comprising this DNA and various regulatory elements (including a promoter, a ribosome binding site, a terminator, an enhancer, and various cis elements that control the expression level) for expressing the amino acid sequence in a host cell. A recombinant vector having the gene construct for use is constructed according to the host cell.
This recombinant vector is introduced into a predetermined host cell (for example, yeast, insect cell, plant cell, animal (mammalian) cell) by a general technique, and the host cell or a tissue or an individual containing the cell under a predetermined condition Is cultured. Thereby, the target polypeptide can be expressed and produced in the cell. The target neuronal differentiation-inducing peptide can be obtained by isolating and purifying the polypeptide from the host cell (in the medium if secreted). This recombinant vector is introduced into a predetermined host cell (for example, yeast, insect cell, plant cell, mammalian cell) by a general technique, and the host cell or a tissue or an individual containing the cell is cultured under a predetermined condition. . Thereby, the target polypeptide can be expressed and produced in the cell. The target neuronal differentiation-inducing peptide can be obtained by isolating and purifying the polypeptide from the host cell (in the medium if secreted).
As a method for constructing a recombinant vector and a method for introducing the constructed recombinant vector into a host cell, a method conventionally used in the field may be employed as it is, and such method itself particularly characterizes the present invention. Since it is not a thing, detailed description is abbreviate | omitted.
例えば、宿主細胞内で効率よく大量に生産させるために融合タンパク質発現システムを利用することができる。すなわち、目的の神経分化誘導ペプチドのアミノ酸配列をコードする遺伝子（ＤＮＡ）を化学合成し、該合成遺伝子を適当な融合タンパク質発現用ベクター（例えばノバジェン社から提供されているｐＥＴシリーズおよびアマシャムバイオサイエンス社から提供されているｐＧＥＸシリーズのようなＧＳＴ(Glutathione S-transferase)融合タンパク質発現用ベクター）の好適なサイトに導入する。そして該ベクターにより宿主細胞（典型的には大腸菌）を形質転換する。得られた形質転換体を培養して目的の融合タンパク質を調製する。次いで、該タンパク質を抽出及び精製する。次いで、得られた精製融合タンパク質を所定の酵素（プロテアーゼ）で切断し、遊離した目的のペプチド断片（設計した神経分化誘導ペプチド）をアフィニティクロマトグラフィー等の方法によって回収する。このような従来公知の融合タンパク質発現システム（例えばアマシャムバイオサイエンス社により提供されるＧＳＴ／Ｈｉｓシステムを利用し得る。）を用いることによって、本発明の神経分化誘導ペプチドを製造することができる。
或いは、無細胞タンパク質合成システム用の鋳型ＤＮＡ（即ち神経分化誘導ペプチドのアミノ酸配列をコードするヌクレオチド配列を含む合成遺伝子断片）を構築し、ペプチド合成に必要な種々の化合物（ＡＴＰ、ＲＮＡポリメラーゼ、アミノ酸類等）を使用し、いわゆる無細胞タンパク質合成システムを採用して目的のポリペプチドをインビトロ合成することができる。無細胞タンパク質合成システムについては、例えばShimizuらの論文(Shimizu et al., Nature Biotechnology, 19, 751-755(2001))、Madinらの論文(Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), 559-564(2000))が参考になる。これら論文に記載された技術に基づいて、本願出願時点において既に多くの企業がポリペプチドの受託生産を行っており、また、無細胞タンパク質合成用キット（例えば、日本の東洋紡績（株）から入手可能なPROTEIOS（商標）Wheat germ cell-free protein synthesis kit）が市販されている。
従って、上述のようにして、利用するアミノ酸配列（ＲＡＣＫ１関連アミノ酸配列）をひとたび決定し、ペプチド鎖を設計しさえすれば、そのアミノ酸配列に従って無細胞タンパク質合成システムによって目的の神経分化誘導ペプチドを容易に合成・生産することができる。例えば、日本の（株）ポストゲノム研究所のピュアシステム（登録商標）に基づいて本発明の神経分化誘導ペプチドを容易に生産することができる。For example, a fusion protein expression system can be used for efficient mass production in a host cell. That is, a gene (DNA) encoding an amino acid sequence of a target neuronal differentiation-inducing peptide is chemically synthesized, and the synthesized gene is converted into an appropriate fusion protein expression vector (for example, the pET series and Amersham Biosciences provided by Novagen) GST (Glutathione S-transferase) fusion protein expression vector) such as pGEX series provided by A host cell (typically E. coli) is transformed with the vector. The obtained transformant is cultured to prepare the desired fusion protein. The protein is then extracted and purified. Next, the obtained purified fusion protein is cleaved with a predetermined enzyme (protease), and the released target peptide fragment (designed neuronal differentiation-inducing peptide) is recovered by a method such as affinity chromatography. By using such a conventionally known fusion protein expression system (for example, the GST / His system provided by Amersham Bioscience) can be used, the neuronal differentiation-inducing peptide of the present invention can be produced.
Alternatively, a template DNA for a cell-free protein synthesis system (that is, a synthetic gene fragment containing a nucleotide sequence encoding the amino acid sequence of a neuronal differentiation-inducing peptide) is constructed, and various compounds (ATP, RNA polymerase, amino acids) required for peptide synthesis are constructed. The desired polypeptide can be synthesized in vitro using a so-called cell-free protein synthesis system. Regarding cell-free protein synthesis systems, for example, Shimizu et al. (Shimizu et al., Nature Biotechnology, 19, 751-755 (2001)), Madin et al. (Madin et al., Proc. Natl. Acad. Sci. USA, 97 (2), 559-564 (2000)) is helpful. Based on the techniques described in these papers, many companies have already commissioned production of polypeptides at the time of filing this application, and also have cell-free protein synthesis kits (for example, obtained from Toyobo Co., Ltd., Japan). PROTEIOS (trademark) Wheat germ cell-free protein synthesis kit) is commercially available.
Therefore, once the amino acid sequence (RACK1-related amino acid sequence) to be used is determined and the peptide chain is designed as described above, the target neuronal differentiation-inducing peptide can be easily achieved by the cell-free protein synthesis system according to the amino acid sequence. Can be synthesized and produced. For example, the neuronal differentiation-inducing peptide of the present invention can be easily produced based on the Pure System (registered trademark) of Post Genome Research Institute, Japan.
ここで開示される神経分化誘導ペプチドをコードするヌクレオチド配列及び／又は該配列と相補的なヌクレオチド配列を含む一本鎖又は二本鎖のポリヌクレオチドは、従来公知の方法によって容易に製造（合成）することができる。すなわち、設計したアミノ酸配列を構成する各アミノ酸残基に対応するコドンを選択することによって、神経分化誘導ペプチドのアミノ酸配列に対応するヌクレオチド配列が容易に決定され、提供される。そして、ひとたびヌクレオチド配列が決定されれば、ＤＮＡ合成機等を利用して、所望するヌクレオチド配列に対応するポリヌクレオチド（一本鎖）を容易に得ることができる。さらに得られた一本鎖ＤＮＡを鋳型として用い、種々の酵素的合成手段（典型的にはＰＣＲ）を採用して目的の二本鎖ＤＮＡを得ることができる。
本発明によって提供されるポリヌクレオチドは、ＤＮＡの形態であってもよく、ＲＮＡ（ｍＲＮＡ等）の形態であってもよい。ＤＮＡは、二本鎖又は一本鎖で提供され得る。一本鎖で提供される場合は、コード鎖（センス鎖）であってもよく、それと相補的な配列の非コード鎖（アンチセンス鎖）であってもよい。
本発明によって提供されるポリヌクレオチドは、上述のように、種々の宿主細胞中で又は無細胞タンパク質合成システムにて、神経分化誘導ペプチド生産のための組換え遺伝子（発現カセット）を構築するための材料として使用することができる。
本発明によると、新規なアミノ酸配列の神経分化誘導ペプチドをコードするヌクレオチド配列及び／又は該配列と相補的なヌクレオチド配列を含むポリヌクレオチドが提供される。例えば、ペプチド鎖を構成する全アミノ酸残基数が５０以下（好ましくは４０以下）であって、配列番号１〜１２で示されるアミノ酸配列或いは該配列の改変配列を含むアミノ酸配列をコードするヌクレオチド配列及び／又は該配列と相補的なヌクレオチド配列を含む（又はそれら配列から実質的に構成された）人為的に設計されたポリヌクレオチドが提供される。A single-stranded or double-stranded polynucleotide comprising a nucleotide sequence encoding the neuronal differentiation-inducing peptide disclosed herein and / or a nucleotide sequence complementary to the sequence is easily produced (synthesized) by a conventionally known method. can do. That is, by selecting a codon corresponding to each amino acid residue constituting the designed amino acid sequence, a nucleotide sequence corresponding to the amino acid sequence of the neuronal differentiation-inducing peptide is easily determined and provided. Once the nucleotide sequence is determined, a polynucleotide (single strand) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesizer or the like. Furthermore, using the obtained single-stranded DNA as a template, various enzymatic synthesis means (typically PCR) can be employed to obtain the desired double-stranded DNA.
The polynucleotide provided by the present invention may be in the form of DNA or RNA (such as mRNA). DNA can be provided as double-stranded or single-stranded. When provided as a single strand, it may be a coding strand (sense strand) or a non-coding strand (antisense strand) having a sequence complementary thereto.
The polynucleotide provided by the present invention is for constructing a recombinant gene (expression cassette) for producing a neuronal differentiation-inducing peptide in various host cells or in a cell-free protein synthesis system as described above. Can be used as material.
According to the present invention, a polynucleotide comprising a nucleotide sequence encoding a neuronal differentiation-inducing peptide having a novel amino acid sequence and / or a nucleotide sequence complementary to the sequence is provided. For example, the total number of amino acid residues constituting the peptide chain is 50 or less (preferably 40 or less), and a nucleotide sequence encoding an amino acid sequence represented by SEQ ID NOs: 1 to 12 or an amino acid sequence containing a modified sequence of the sequence And / or artificially designed polynucleotides comprising (or consisting essentially of) nucleotide sequences complementary to the sequences are provided.
好適な本発明の神経分化誘導ペプチドは少なくとも一種の細胞に対して高い神経分化誘導活性を有する。このため、神経分化誘導剤の有効成分として好適に使用し得る。なお、神経分化誘導剤に含有される神経分化誘導ペプチドは、神経分化誘導活性を損なわない限りにおいて、塩の形態であってもよい。例えば、常法に従って通常使用されている無機酸又は有機酸を付加反応させることにより得られ得る該ペプチドの酸付加塩を使用することができる。或いは、神経分化誘導活性を有する限り、他の塩（例えば金属塩）であってもよい。 A preferred neuronal differentiation-inducing peptide of the present invention has a high neuronal differentiation-inducing activity against at least one cell. For this reason, it can be suitably used as an active ingredient of a neuronal differentiation inducer. The neuronal differentiation-inducing peptide contained in the neuronal differentiation-inducing agent may be in the form of a salt as long as the neuronal differentiation-inducing activity is not impaired. For example, an acid addition salt of the peptide that can be obtained by addition reaction of an inorganic acid or an organic acid usually used according to a conventional method can be used. Alternatively, other salts (for example, metal salts) may be used as long as they have nerve differentiation-inducing activity.
神経分化誘導剤は、有効成分である神経分化誘導ペプチドの他、使用形態に応じて薬学（医薬）上許容され得る種々の担体を含み得る。希釈剤、賦形剤等としてペプチド医薬において一般的に使用される担体が好ましい。神経分化誘導剤の用途や形態に応じて適宜異なり得るが、典型的には、水、生理学的緩衝液、種々の有機溶媒が挙げられる。適当な濃度のアルコール（エタノール等）水溶液、グリセロール、オリーブ油のような不乾性油であり得る。或いはリポソームであってもよい。また、神経分化誘導剤に含有させ得る副次的成分としては、種々の充填剤、増量剤、結合剤、付湿剤、表面活性剤、色素、香料等が挙げられる。
神経分化誘導剤の形態に関して特に限定はない。例えば、典型的な形態として、液剤、懸濁剤、乳剤、エアロゾル、泡沫剤、顆粒剤、粉末剤、錠剤、カプセル、軟膏が挙げられる。また、注射等に用いるため、使用直前に生理食塩水又は適当な緩衝液（例えばＰＢＳ）等に溶解して薬液を調製するための凍結乾燥物、造粒物とすることもできる。
なお、神経分化誘導ペプチド（主成分）及び種々の担体（副成分）を材料にして種々の形態の薬剤（組成物）を調製するプロセス自体は従来公知の方法に準じればよく、かかる製剤方法自体は本発明を特徴付けるものでもないため詳細な説明は省略する。処方に関する詳細な情報源として、例えばComprehensive Medicinal Chemistry, Corwin Hansch監修，Pergamon Press刊(1990)が挙げられる。この書籍の全内容は本明細書中に参照として援用されている。The nerve differentiation-inducing agent can contain various carriers that are pharmaceutically (pharmaceutical) acceptable depending on the form of use in addition to the neuronal differentiation-inducing peptide that is an active ingredient. Carriers generally used in peptide medicine as diluents, excipients and the like are preferred. Typically, water, a physiological buffer solution, and various organic solvents can be mentioned, although it may vary depending on the use and form of the neuronal differentiation inducer. It can be a non-drying oil such as an aqueous solution of alcohol (such as ethanol) of a suitable concentration, glycerol, olive oil. Or a liposome may be sufficient. Moreover, as a secondary component which can be contained in a nerve differentiation-inducing agent, various fillers, extenders, binders, moisturizers, surfactants, pigments, fragrances and the like can be mentioned.
There is no particular limitation regarding the form of the neuronal differentiation inducer. For example, typical forms include solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules and ointments. Moreover, since it uses for injection etc., it can also be set as the freeze-dried material and granulated material for melt | dissolving in a physiological saline or a suitable buffer solution (for example, PBS) etc. just before use, and preparing a chemical | medical solution.
In addition, the process itself for preparing various forms of drugs (compositions) using a neuronal differentiation-inducing peptide (main component) and various carriers (subcomponents) as materials may be in accordance with a conventionally known method. As such, it does not characterize the present invention and will not be described in detail. As a detailed information source on prescription, for example, Comprehensive Medicinal Chemistry, supervised by Corwin Hansch, published by Pergamon Press (1990) can be mentioned. The entire contents of this book are incorporated herein by reference.
本発明によって提供される神経分化誘導剤は、その形態及び目的に応じた方法や用量で使用することができる。
例えば、ここで開示されるＲＡＣＫ１関連アミノ酸配列を含む神経分化誘導ペプチド(即ち該ペプチドを含む神経分化誘導剤は、液剤として、静脈内、筋肉内、皮下、皮内若しくは腹腔内への注射によって患者(即ち生体)に所望する量だけ投与することができる。或いは、錠剤等の固体形態のものは経口投与することができる。これにより、生体内で、典型的には患部又はその周辺に存在する体性幹細胞から、神経細胞を発生(生産)させることができる。このため、神経再生が有力な治療法となる種々の神経疾患を効果的に治療することが可能となる。例えば、パーキンソン病、脳梗塞、アルツハイマー病、脊髄損傷による身体の麻痺、脳挫傷、筋萎縮性側索硬化症、ハンチントン病、脳腫瘍、網膜変性症等の神経疾患を再生医療的アプローチによって治療することが実現される。The nerve differentiation-inducing agent provided by the present invention can be used in a method or dosage depending on its form and purpose.
For example, a neuronal differentiation-inducing peptide comprising the RACK1-related amino acid sequence disclosed herein (ie, a neuronal differentiation-inducing agent comprising the peptide is administered as a solution by intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal injection. (Ie, a living body) can be administered in a desired amount, or a solid form such as a tablet can be administered orally, so that it is present in a living body, typically in or around the affected area. Nerve cells can be generated (produced) from somatic stem cells, so that it is possible to effectively treat various neurological diseases that are effective treatments for nerve regeneration, such as Parkinson's disease, Regenerative medical approach to neurological diseases such as cerebral infarction, Alzheimer's disease, body paralysis due to spinal cord injury, brain contusion, amyotrophic lateral sclerosis, Huntington's disease, brain tumor, retinal degeneration Thus treating is realized.
或いはまた、生体から一時的に又は永久的に摘出した細胞材料、即ち生組織や細胞塊(例えば体性幹細胞の培養物)に、適当量の神経分化誘導剤（神経分化誘導ペプチド）を付与することによって、体外(インビトロ)で神経細胞を効率よく発生させることができる。このことは当該細胞材料中に所望する神経細胞を大量に生産し得ることを意味する。
而して、大量に生産された神経細胞、或いは該生産された神経細胞を含む細胞材料（生組織や細胞塊）を再び生体内(典型的には神経再生が要求されている患部）に戻すことによっても、生体に直接神経分化誘導剤（神経分化誘導ペプチド）を投与する場合と同様の治療効果が得られ得る。
以上の説明から明らかなように、本発明はまた、ここで開示される神経分化誘導ペプチドのいずれかを利用することによって、神経疾患治療に有用な、神経細胞に分化誘導された細胞、細胞塊又は生組織を提供することができる。Alternatively, an appropriate amount of neuronal differentiation-inducing agent (neural differentiation-inducing peptide) is applied to cell material temporarily or permanently removed from a living body, that is, a living tissue or a cell mass (for example, a culture of somatic stem cells). As a result, nerve cells can be efficiently generated in vitro (in vitro). This means that the desired nerve cells can be produced in large quantities in the cell material.
Thus, nerve cells produced in large quantities or cell materials (living tissue or cell mass) containing the produced nerve cells are returned to the living body (typically, the affected area where nerve regeneration is required). In this way, the same therapeutic effect as when a nerve differentiation-inducing agent (nerve differentiation-inducing peptide) is directly administered to a living body can be obtained.
As is clear from the above description, the present invention also provides cells, cell masses induced to differentiate into nerve cells, which are useful for treating neurological diseases by using any of the neuronal differentiation-inducing peptides disclosed herein. Alternatively, live tissue can be provided.
また、本発明の神経分化誘導ペプチドをコードするポリヌクレオチドは、いわゆる遺伝子治療に使用する素材として用い得る。例えば、神経分化誘導ペプチドをコードする遺伝子（典型的にはＤＮＡセグメント、或いはＲＮＡセグメント）を適当なベクターに組み込み、目的とする部位に導入することにより、常時、生体（細胞）内で本発明に係る神経分化誘導ペプチドを発現させることが可能である。従って、本発明の神経分化誘導ペプチドをコードするポリヌクレオチド（ＤＮＡセグメント、ＲＮＡセグメント等）は、上述した患者等に対し、神経疾患を治療し又は予防する薬剤として有用である。 The polynucleotide encoding the neuronal differentiation-inducing peptide of the present invention can be used as a material used for so-called gene therapy. For example, a gene encoding a neuronal differentiation-inducing peptide (typically a DNA segment or an RNA segment) is incorporated into an appropriate vector and introduced into a target site, so that the present invention is always in vivo (cell). Such neuronal differentiation-inducing peptides can be expressed. Therefore, the polynucleotide (DNA segment, RNA segment, etc.) encoding the neuronal differentiation-inducing peptide of the present invention is useful as a drug for treating or preventing neurological diseases for the above-mentioned patients.
以下、本発明に関するいくつかの実施例を説明するが、本発明をかかる実施例に示すものに限定することを意図したものではない。 Several examples relating to the present invention will be described below, but the present invention is not intended to be limited to those shown in the examples.
＜実施例１：ペプチド合成＞
計４種類のペプチド（サンプル１〜４）を後述するペプチド合成機を用いて製造した。表１には、これら合成ペプチドのアミノ酸配列を列挙している。<Example 1: Peptide synthesis>
A total of four types of peptides (samples 1 to 4) were produced using the peptide synthesizer described below. Table 1 lists the amino acid sequences of these synthetic peptides.
表１に示すように、サンプル１〜４は、いずれもＮ末端側にＲＡＣＫ１関連アミノ酸配列（配列番号２〜５参照）を有し、そのＣ末端側に隣接して核小体局在シグナル関連アミノ酸配列（配列番号１参照）を有している。なお、いずれのサンプルも、Ｃ末端アミノ酸のカルボキシル基（−ＣＯＯＨ）はアミド化（−ＣＯＮＨ２）されている。As shown in Table 1, each of samples 1 to 4 has a RACK1-related amino acid sequence (see SEQ ID NOs: 2 to 5) on the N-terminal side, and is associated with a nucleolar localization signal adjacent to the C-terminal side. It has an amino acid sequence (see SEQ ID NO: 1). In any sample, the carboxyl group (—COOH) of the C-terminal amino acid is amidated (—CONH 2 ).
上述した各ペプチドは、市販のペプチド合成機（Intavis AG社製品 Multi Pep RS）を用いて固相合成法（Ｆｍｏｃ法）により合成した。なお、縮合剤としてＨＡＴＵ（O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluoro-phosphate、渡辺化学工業（株）製品）およびＤＩＥＡ（ジイソプロピルエチルアミン、和光純薬工業（株）製品）を使用し、固相合成法に用いた樹脂及びアミノ酸はNOVA biochem社から購入した。アミノ酸配列のＣ末端をアミド化する場合には、固相担体として「Rink Amide resin (100〜200 mesh)」 を使用した。
而して、上記ペプチド合成機の合成プログラムに準じて脱保護基反応及び縮合反応を反復して樹脂に結合するFmoc−アミノ酸からペプチド鎖を伸長していき、目的の鎖長の合成ペプチドを得た。具体的には、２０％ピペリジン／ジメチルホルムアミド(DMF)（ペプチド合成用グレード、和光純薬工業（株）製品）によって、アミノ酸のアミノ保護基であるFmocを切断除去し、ＤＭＦで洗浄し、Fmoc−アミノ酸(-OH)各４ｅｑを反応させ、ＤＭＦで洗浄する操作を反復した。そして、ペプチド鎖の伸長反応が全て終了した後、２０％ピペリジン／ＤＭＦによりFmoc基を切断し、ＤＭＦ、エタノールの順で上記反応物を洗浄した。Each peptide described above was synthesized by a solid phase synthesis method (Fmoc method) using a commercially available peptide synthesizer (product of Intavis AG, Multi Pep RS). As condensation agents, HATU (O- (7-azabenzotriazol-l-yl) -l, l, 3,3-tetramethyluronium hexafluoro-phosphate, product of Watanabe Chemical Co., Ltd.) and DIEA (diisopropylethylamine, Wako Pure Chemical Industries, Ltd.) The resin and amino acids used in the solid phase synthesis method were purchased from NOVA biochem. When amidating the C-terminal of the amino acid sequence, “Rink Amide resin (100 to 200 mesh)” was used as a solid support.
Thus, according to the synthesis program of the above peptide synthesizer, the deprotection group reaction and the condensation reaction are repeated to extend the peptide chain from the Fmoc-amino acid that binds to the resin to obtain a synthetic peptide having the desired chain length. It was. Specifically, 20% piperidine / dimethylformamide (DMF) (grade for peptide synthesis, product of Wako Pure Chemical Industries, Ltd.) was used to cleave off Fmoc, which is an amino protecting group of amino acid, and washed with DMF. -The operation of reacting 4 eq of each amino acid (-OH) and washing with DMF was repeated. After the peptide chain elongation reaction was completed, the Fmoc group was cleaved with 20% piperidine / DMF, and the reaction product was washed with DMF and ethanol in this order.
固相合成後、合成したペプチド鎖を樹脂と共に遠沈管に移し、エタンジオール１．８ｍＬ、m-クレゾール０．６ｍＬ、チオアニソール３．６ｍＬ及びトリフルオロ酢酸２４ｍＬを加え、室温で２時間撹拌した。その後、ペプチド鎖に結合していた樹脂を濾過して除去した。
次いで、濾液に冷ジエチルエーテルを加え、氷冷水で冷却してペプチド沈澱物を得た。その後、遠心分離（2500rpmで５分間）によって上澄みを廃棄した。沈殿物に冷ジエチルエーテルを新たに加えて十分に撹拌した後、上記と同じ条件で遠心分離を行った。この撹拌と遠心分離の処理を計３回反復して行った。After the solid phase synthesis, the synthesized peptide chain was transferred to a centrifuge tube together with the resin, ethanediol 1.8 mL, m-cresol 0.6 mL, thioanisole 3.6 mL, and trifluoroacetic acid 24 mL were added, and the mixture was stirred at room temperature for 2 hours. Thereafter, the resin bound to the peptide chain was removed by filtration.
Next, cold diethyl ether was added to the filtrate and cooled with ice-cold water to obtain a peptide precipitate. Thereafter, the supernatant was discarded by centrifugation (2500 rpm for 5 minutes). Cold diethyl ether was newly added to the precipitate and sufficiently stirred, and then centrifuged under the same conditions as described above. This stirring and centrifugation process was repeated three times in total.
得られたペプチド沈殿物を真空乾燥し、高速液体クロマトグラフ（Waters 600：Waters社製品）を用いて精製を行った。
具体的には、プレカラム（日本ウォーターズ（株）製品、Guard-Pak Delta-pak C18 A300）及びＣ１８逆相カラム（日本ウォーターズ（株）製品、XTerra（登録商標）カラム、MS C18、5μm、4.6×150mm）を使用し、０．１％トリフルオロ酢酸水溶液と０．１％トリフルオロ酢酸アセトニトリル溶液との混合液を溶離液に用いた。即ち、溶離液に含まれる上記トリフルオロ酢酸アセトニトリル溶液の分量を経時的に増大させつつ（容積比で１０％から８０％への濃度勾配を設ける）、１．５ｍＬ／分の流速で上記カラムを用いて３０〜４０分間の分離精製を行った。なお、逆相カラムから溶離したペプチドは紫外線検出器（490E Detector：Waters社製品）を用いて波長：２２０ｎｍで検出され、記録チャート上にピークとして示される。
また、溶離した各ペプチドの分子量をPerSeptive Biosystems社製のVoyager DE RP（商標）を用いてMALDI-TOF/MS（Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry：マトリックス支援レーザーイオン化−飛行時間型−質量分析)に基づいて決定した。その結果、目的のペプチドが合成・精製されていることが確認された。The obtained peptide precipitate was vacuum-dried and purified using a high performance liquid chromatograph (Waters 600: product of Waters).
Specifically, pre-column (Nippon Waters Co., Ltd. product, Guard-Pak Delta-pak C18 A300) and C18 reverse phase column (Nippon Waters Co., Ltd. product, XTerra (registered trademark) column, MS C18, 5 μm, 4.6 × 150 mm), and a mixed solution of 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution was used as an eluent. That is, while increasing the amount of the trifluoroacetic acid acetonitrile solution contained in the eluent over time (providing a concentration gradient from 10% to 80% in volume ratio), the column was moved at a flow rate of 1.5 mL / min. Separation and purification was performed for 30 to 40 minutes. The peptide eluted from the reverse phase column is detected at a wavelength of 220 nm using an ultraviolet detector (490E Detector: Waters product), and is shown as a peak on the recording chart.
In addition, the molecular weight of each eluted peptide was determined using MALDI-TOF / MS (Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry) using Voyager DE RP (trademark) manufactured by PerSeptive Biosystems. Analysis). As a result, it was confirmed that the target peptide was synthesized and purified.
＜実施例２：合成ペプチドの神経分化誘導活性評価＞
上記実施例１で得られた神経分化誘導ペプチド（サンプル１〜４）のそれぞれについて神経分化誘導活性を調べた。
即ち、サンプルペプチドを、マウスから採取した神経幹細胞の培養液（マウス神経幹細胞増殖培地：Cell Applications社製品）中に添加し、インキュベーションした。添加濃度は、何れのペプチドについても１μＭとした。
そして、ペプチドを添加して７日間経過後、各培養細胞について、ＤＡＰＩ（4',6-diamidino-2-phenylindole）による核染色を行い、蛍光顕微鏡で観察した。また、同じサンプルに対して神経分化誘導マーカーによる評価を行った。即ち、ニューロン（神経細胞）を識別するマーカーとしてチューブリン（具体的にはβ３−チューブリン）を採用し、当該チューブリンを識別する蛍光色素標識抗チューブリン抗体を用いた蛍光抗体法によって培養液中のチューブリンの存在（即ちニューロンの存在）の有無を確認した。結果を図１〜図３に示す。これら図面は、各サンプルペプチドを添加して７日間培養した後のマウス神経幹細胞の神経分化の状態を調べた蛍光顕微鏡写真（画像）であり、ＤＩＣ画像と、ＤＡＰＩによる核染色画像と、蛍光色素標識抗チューブリン抗体を使用した免疫抗体法で調べた結果を示す蛍光画像とを重ねた（マージした）画像である。図１はサンプル１を添加した培養細胞の結果を示し、図２はサンプル３を添加した培養細胞の結果を示し、図３はサンプル４を添加した培養細胞の結果を示す。
なお、ポジティブコントロールとして、いずれのペプチドも添加することなく、マウス神経幹細胞をマウス神経幹細胞分化培地（Cell Applications社製品）で培養し、培養開始から７日間経過した後に上記と同様の処理及び蛍光顕微鏡観察を行った。結果を図４に示す。
また、ネガティブコントロールとして、いずれのペプチドも添加することなく、マウス神経幹細胞をマウス神経幹細胞増殖培地（Cell Applications社製品）で培養し、培養開始から７日間経過した後に上記と同様の処理及び蛍光顕微鏡観察を行った。結果を図５に示す。<Example 2: Evaluation of nerve differentiation-inducing activity of synthetic peptide>
The neuronal differentiation-inducing activity of each of the neuronal differentiation-inducing peptides (samples 1 to 4) obtained in Example 1 was examined.
That is, the sample peptide was added to a culture solution of neural stem cells collected from mice (mouse neural stem cell growth medium: Cell Applications product) and incubated. The addition concentration was 1 μM for all peptides.
After 7 days from the addition of the peptide, each cultured cell was subjected to nuclear staining with DAPI (4 ′, 6-diamidino-2-phenylindole) and observed with a fluorescence microscope. In addition, the same sample was evaluated with a neuronal differentiation-inducing marker. That is, tubulin (specifically β3-tubulin) is employed as a marker for identifying neurons (neural cells), and the culture solution is obtained by a fluorescent antibody method using a fluorescent dye-labeled anti-tubulin antibody for identifying the tubulin. The presence or absence of tubulin (ie, the presence of neurons) was confirmed. The results are shown in FIGS. These drawings are fluorescent micrographs (images) of the state of neuronal differentiation of mouse neural stem cells after each sample peptide was added and cultured for 7 days. DIC image, nuclear staining image by DAPI, fluorescent dye It is the image which overlap | superposed (merged) with the fluorescence image which shows the result investigated by the immunity antibody method using labeled anti-tubulin antibody. FIG. 1 shows the results of cultured cells to which sample 1 was added, FIG. 2 shows the results of cultured cells to which sample 3 was added, and FIG. 3 shows the results of cultured cells to which sample 4 was added.
As a positive control, mouse neural stem cells were cultured in mouse neural stem cell differentiation medium (product of Cell Applications) without adding any peptide, and after 7 days from the start of culture, the same treatment and fluorescence microscope as above Observations were made. The results are shown in FIG.
As a negative control, mouse neural stem cells were cultured in mouse neural stem cell growth medium (product of Cell Applications) without adding any peptide, and after 7 days from the start of culture, the same treatment and fluorescence microscope as described above Observations were made. The results are shown in FIG.
上記評価試験の結果、サンプル１〜４の人工ペプチド（神経分化誘導ペプチド）を添加した場合（図１〜３参照）、ポジティブコントロール（図４）以上の顕著な神経分化が認められた。即ち、サンプル１〜４のいずれのペプチドを添加した場合でも、蛍光色素標識抗チューブリン抗体の存在による蛍光発色が良好に認められた。同じ増殖培地を使用したネガティブコントロールでは、このような蛍光発色（神経分化）は認められなかった（図５）。
このことは、サンプルペプチドの添加によって、神経幹細胞がニューロンに分化したことを示している。As a result of the evaluation test, when the artificial peptides (neural differentiation-inducing peptides) of Samples 1 to 4 were added (see FIGS. 1 to 3), remarkable neuronal differentiation higher than the positive control (FIG. 4) was observed. That is, even when any of the peptides of Samples 1 to 4 was added, good fluorescence was observed due to the presence of the fluorescent dye-labeled anti-tubulin antibody. In the negative control using the same growth medium, such fluorescent color development (neural differentiation) was not observed (FIG. 5).
This indicates that neural stem cells differentiated into neurons by the addition of the sample peptide.
＜実施例３：顆粒剤の調製＞
サンプル１のペプチド５０ｍｇと結晶化セルロース５０ｍｇ及び乳糖４００ｍｇとを混合した後、エタノールと水の混合液１ｍＬを加え混練した。この混練物を常法に従って造粒し、神経分化誘導ペプチドを主成分とする顆粒剤（顆粒状神経分化誘導剤）を得た。<Example 3: Preparation of granules>
After mixing 50 mg of the peptide of sample 1, 50 mg of crystallized cellulose and 400 mg of lactose, 1 mL of a mixed solution of ethanol and water was added and kneaded. This kneaded product was granulated according to a conventional method to obtain a granule (granular neuronal differentiation inducer) containing a neuronal differentiation-inducing peptide as a main component.
上述のように本発明の神経分化誘導ペプチドは高い神経分化誘導活性を有しているため、医薬用のペプチド成分として利用することができる。 As described above, since the neuronal differentiation-inducing peptide of the present invention has a high neuronal differentiation-inducing activity, it can be used as a pharmaceutical peptide component.
配列番号１〜配列番号１２ 合成ペプチド SEQ ID NO: 1 to SEQ ID NO: 12 synthetic peptide
Claims (10)
ＲＡＣＫ１関連アミノ酸配列として以下のアミノ酸配列：
Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-（Ｉ又はＶ）-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ
を含む人為的に合成されたペプチドと、
薬学上許容され得る少なくとも一種の担体と、
を含有し、
ここで、前記ペプチドを構成する全アミノ酸残基数が３５以下である、神経分化誘導剤。 A neuronal differentiation inducer capable of inducing differentiation of at least one stem cell into a neuronal cell,
The following amino acid sequences as RACK1-related amino acid sequences:
T-L-D-G-G-D- (I or V) -I-N-A-L-C-F-S
An artificially synthesized peptide containing
At least one pharmaceutically acceptable carrier;
Contain,
Here, the neuronal differentiation inducing agent , wherein the total number of amino acid residues constituting the peptide is 35 or less .
Ｋ-Ｋ-Ｒ-Ｔ-Ｌ-Ｒ-Ｋ-Ｎ-Ｄ-Ｒ-Ｋ-Ｋ-Ｒ（配列番号１）
をさらに含む人為的に合成されたペプチドである、請求項１に記載の神経分化誘導剤。 The peptide comprises the following amino acid sequences that constitute the nucleolar localization signal:
KK-R-T-L-R-K-N-D-R-K-K-R (SEQ ID NO: 1)
The neuronal differentiation-inducing agent according to claim 1, wherein the agent is an artificially synthesized peptide.
Ｌ-Ｙ-Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-（Ｉ又はＶ）-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ-Ｐ-Ｎ-Ｒ-Ｙ-Ｗ-Ｌ
を含む、請求項１または２に記載の神経分化誘導剤。 The peptide has the following amino acid sequence as the RACK1-related amino acid sequence:
L-Y-T-L-D-G-G-D- (I or V) -I-N-A-L-C-F-S-P-N-R-Y-W-L
The nerve differentiation-inducing agent according to claim 1 or 2 , comprising
Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-（Ｉ又はＶ）-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ
を含み、且つ、核小体局在シグナルを構成する以下のアミノ酸配列：
Ｋ-Ｋ-Ｒ-Ｔ-Ｌ-Ｒ-Ｋ-Ｎ-Ｄ-Ｒ-Ｋ-Ｋ-Ｒ（配列番号１）
をさらに含み、該ペプチドを構成する全アミノ酸残基数が３５以下である、ペプチド。 An artificially synthesized peptide capable of inducing differentiation of at least one kind of stem cell into a neuron, and the following amino acid sequence as a RACK1-related amino acid sequence:
T-L-D-G-G-D- (I or V) -I-N-A-L-C-F-S
And the following amino acid sequences constituting a nucleolar localization signal:
KK-R-T-L-R-K-N-D-R-K-K-R (SEQ ID NO: 1)
Further seen including, total number of amino acid residues constituting the peptide is 35 or less, a peptide.
Ｌ-Ｙ-Ｔ-Ｌ-Ｄ-Ｇ-Ｇ-Ｄ-（Ｉ又はＶ）-Ｉ-Ｎ-Ａ-Ｌ-Ｃ-Ｆ-Ｓ-Ｐ-Ｎ-Ｒ-Ｙ-Ｗ-Ｌ
を含む、請求項６に記載のペプチド。 As the RACK1-related amino acid sequence, the following amino acid sequence:
L-Y-T-L-D-G-G-D- (I or V) -I-N-A-L-C-F-S-P-N-R-Y-W-L
The peptide according to claim 6 , comprising:
請求項１〜５のいずれかに記載の神経分化誘導剤または請求項６〜９のいずれかに記載のペプチドを前記細胞材料に供給することを特徴とする神経細胞の生産方法。 A method for producing nerve cells from at least one cell material,
A method for producing nerve cells, comprising supplying the cell material with the nerve differentiation-inducing agent according to any one of claims 1 to 5 or the peptide according to any one of claims 6 to 9 .
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| Title |
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| JPN6010038849; ＬＩＵ ＹＶ．， ｅｔ ａｌ．: 'ＲＡＣＫ１ ｃｏｍｐｅｔｅｓ ｗｉｔｈ ＨＳＰ９０ ｆｏｒ ｂｉｎｄｉｎｇ ｔｏ ＨＩＦ-１ａｌｐｈａ ａｎｄ ｉｓ ｒｅｑｕｉｒｅｄ ｆｏｒ Ｏ（２）-ｉｎｄｅｐｅｎｄｅｎｔ ａｎｄ ＨＳＰ９０ ｉ' Ｍｏｌ． Ｃｅｌｌ ｖｏｌ．２５， ｎｏ．２, 2007, ｐ．２０７-２１７ * |
| JPN6010038853; ＳＫＬＡＮ ＥＨ．， ｅｔ ａｌ．: 'ＲＡＣＫ１ ｈａｓ ｔｈｅ ｎｅｒｖｅ ｔｏ ａｃｔ： ｓｔｒｕｃｔｕｒｅ ｍｅｅｔｓ ｆｕｎｃｔｉｏｎ ｉｎ ｔｈｅ ｎｅｒｖｏｕｓ ｓｙｓｔｅｍ' Ｐｒｏｇ． Ｎｅｕｒｏｂｉｏｌ． ｖｏｌ．７８， ｎｏ．２, 2006, ｐ．１１７-１３４ * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2010117078A1 (en) | 2010-10-14 |
| JPWO2010117078A1 (en) | 2012-10-18 |
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