JPH10276783A - Protein for accelerating hydrolysis of gtp - Google Patents
Protein for accelerating hydrolysis of gtpInfo
- Publication number
- JPH10276783A JPH10276783A JP9090706A JP9070697A JPH10276783A JP H10276783 A JPH10276783 A JP H10276783A JP 9090706 A JP9090706 A JP 9090706A JP 9070697 A JP9070697 A JP 9070697A JP H10276783 A JPH10276783 A JP H10276783A
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- Japan
- Prior art keywords
- leu
- glu
- ser
- lys
- ala
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、細胞内小胞輸送に
関与するRab3サブファミリーメンバーに特異的に作用す
る制御因子、該因子をコードするDNA、該DNAを含むベク
ター、該ベクターを保持する形質転換体、該形質転換体
により産生される組み換えタンパク質に関する。TECHNICAL FIELD The present invention relates to a regulatory factor that specifically acts on a Rab3 subfamily member involved in intracellular vesicle transport, a DNA encoding the factor, a vector containing the DNA, and a vector carrying the vector. The present invention relates to a transformant and a recombinant protein produced by the transformant.
【０００２】[0002]
【従来の技術】Rab（Ras-like proteins in rat brai
n）スモールGタンパク質ファミリーは哺乳動物において
約30種及び酵母において約10種のメンバーからなり、ド
ナー膜からの小胞の出芽、小胞のアクセプター膜との結
合及び融合を含む細胞内小胞輸送に関係している（Taka
i,Y., et al.(1992) Int.Rev.Cytol.133,187-230、Taka
i,Y., et al.(1993) Ciba Foundation Symposium on th
e GTPase superfamily 128-146、Simons,K. and Zeria
l,M.(1993) Neuron 11,789-799、Novick,P. and Breenw
ald,P.(1993) Cell 75,597-601、Nuoffer,C. asnd Balc
h,W.E.(1994) Annu.Rev.Biochem.63,949-990、Pfeffer,
S.R.(1994) Curr.Opin.Cell Biol.6,522-526、Takai,
Y., et al.(1996) Genes To Cells 1,615-632）。いく
つかのRabファミリーメンバーは高相同性膜をもつサブ
ファミリーを構成している。RabファミリーメンバーはG
DP結合非活性形態及びGTP結合活性形態の2つの形態間を
循環し、これに応じて細胞質ゾル及び膜部位間を循環す
る。これら循環する2つの型は小胞輸送におけるこれら
メンバーの作用に必須である(Takai,Y., et al.(1992)
Int.Rev.Cytol.133,187-230、Takai,Y., et al.(1993)
Ciba Foundation Symposium on the GTPase superfamil
y 128-146、Simons,K. and Zerial,M.(1993) Neuron 1
1,789-799、Novick,P. and Breenwald,P.(1993) Cell 7
5,597-601、Nuoffer,C. asnd Balch,W.E.(1994) Annu.R
ev.Biochem.63,949-990、Pfeffer,S.R.(1994)Curr.Opi
n.Cell Biol.6,522-526、Takai,Y., et al.(1996) Gene
s To Cells 1,615-632)。非活性なGDP結合形態は「Rab
GDI」と結合して、細胞質ゾル中に存在する。この形態
は「Rab GEP」の作用により活性なGTP-結合形態へと変
化する。小胞の出芽過程においては、GTP結合活性形態
はドナー膜と相互に作用し小胞の出芽を引き起こす。出
芽の前、途中、または後に、GTP結合形態は「Rab GAP」
の作用によりGDP結合非活性形態へ変化する。小胞がア
クセプター膜に結合する過程においては、GTP結合活性
形態は小胞と相互に作用し、小胞をアクセプター膜へと
転移させる。小胞のアクセプター膜への融合の前、途
中、または後に、GTP結合活性形態は「Rab GAP」の作用
によりGDP結合非活性形態へ変化する。RabはGDP結合非
活性形態に変化すると、再び「Rab GDI」と結合し、細
胞質ゾルへと戻る。[Prior Art] Rab (Ras-like proteins in rat brai
n) The small G protein family consists of about 30 members in mammals and about 10 members in yeast, and intracellular vesicle transport including budding of vesicles from donor membranes, binding and fusion of vesicles to acceptor membranes. Related to (Taka
i, Y., et al. (1992) Int. Rev. Cytol. 133, 187-230, Taka
i, Y., et al. (1993) Ciba Foundation Symposium on th
e GTPase superfamily 128-146, Simons, K. and Zeria
l, M. (1993) Neuron 11,789-799, Novick, P. and Breenw
ald, P. (1993) Cell 75,597-601, Nuoffer, C. asnd Balc
h, WE (1994) Annu. Rev. Biochem. 63, 949-990, Pfeffer,
SR (1994) Curr.Opin.Cell Biol. 6,522-526, Takai,
Y., et al. (1996) Genes To Cells 1,615-632). Several Rab family members constitute a subfamily with highly homologous membranes. Rab family members are G
It circulates between two forms, the DP-bound inactive form and the GTP-bound active form, and accordingly circulates between the cytosol and membrane sites. These two circulating forms are essential for the action of these members in vesicle transport (Takai, Y., et al. (1992)
Int. Rev. Cytol. 133, 187-230, Takai, Y., et al. (1993)
Ciba Foundation Symposium on the GTPase superfamil
y 128-146, Simons, K. and Zerial, M. (1993) Neuron 1
1,789-799, Novick, P. and Breenwald, P. (1993) Cell 7
5,597-601, Nuoffer, C. asnd Balch, WE (1994) Annu.R
ev. Biochem. 63,949-990, Pfeffer, SR (1994) Curr.Opi.
n.Cell Biol. 6,522-526, Takai, Y., et al. (1996) Gene
s To Cells 1,615-632). The inactive form of GDP binding is "Rab
It is present in the cytosol in association with GDI. This form is changed to an active GTP-binding form by the action of "Rab GEP". In the process of vesicle budding, the GTP-binding form interacts with the donor membrane and causes vesicle budding. Before, during or after budding, the GTP-binding form is “Rab GAP”
Changes to an inactive form of GDP binding. In the process of vesicle binding to the acceptor membrane, the active form of GTP binding interacts with the vesicle and transfers the vesicle to the acceptor membrane. Before, during, or after fusion of the vesicle to the acceptor membrane, the GTP binding active form is changed to a GDP binding inactive form by the action of “Rab GAP”. When Rab changes to a GDP-bound inactive form, it binds again to “Rab GDI” and returns to the cytosol.
【０００３】Rabファミリーメンバーに対するこれら3つ
のタイプの制御因子の中で、「RabGDI」は実験が行われ
たすべてのRabファミリーメンバーに作用することが知
られている(Ssasaki,T., et al.(1991) Mol.Cell Biol.
11,2909-2912、Garrett,M.D., et al.(1993) FEBS Let
t.331,233-238、Soldati,T., et al.(1993) Mol.Biol.C
ell 4,425-434、Ullrich,O., et al.(1993) J.Biol.Che
m.268,18143-18150、Beranger,F., et al.(1994) J.Bio
l.Chem.269,13637-13643)。一方、「GAP」に関しては、
酵母のRabファミリーメンバーのうち、「Ypt1」および
「Sec4」には作用せず、「Ypt6」及び「Ypt7」に作用す
る1つのGAPが単離されている(Storm,M.,et al.(1993) N
ature 361,736-739)。しかし、Rabファミリーメンバー
またはRabサブファミリーに特異的なGAPは哺乳動物にお
いて同定されていない。また、「GEP」に関しても、Rab
ファミリーメンバーまたはRabサブファミリーに特異的
なGEPは酵母及び哺乳動物において同定されていない。[0003] Among these three types of regulators for Rab family members, "RabGDI" is known to act on all Rab family members in which experiments were performed (Ssasaki, T., et al. (1991) Mol. Cell Biol.
11,2909-2912, Garrett, MD, et al. (1993) FEBS Let
t.331, 233-238, Soldati, T., et al. (1993) Mol. Biol. C
ell 4,425-434, Ullrich, O., et al. (1993) J. Biol. Che
m.268, 18143-18150, Berganger, F., et al. (1994) J. Bio
l. Chem. 269,13637-13643). On the other hand, for "GAP"
Among the yeast Rab family members, one GAP that does not act on `` Ypt1 '' and `` Sec4 '' but acts on `` Ypt6 '' and `` Ypt7 '' has been isolated (Storm, M., et al. 1993) N
361,736-739). However, no GAP specific for a Rab family member or Rab subfamily has been identified in mammals. Regarding “GEP”, Rab
GEPs specific for family members or the Rab subfamily have not been identified in yeast and mammals.
【０００４】ところで、Rabサブファミリーのうち、Rab
3サブファミリーは、「Rab3A」,「Rab3B」,「Rab3C」,
及び「Rab3D」の4のメンバーから構成されている(Pfeff
er,S.R.(1994) Curr.Opin.Cell Biol.6,522-526)。これ
らのメンバーの中で、「Rab3A」及び「Rab3C」はCa2+依
存性エキソサイトーシスに関係し、特に神経末端からの
神経伝達物質放出という脊椎動物にとって極めて重要な
機能に関係している。一方、「Rab3B」は下垂体細胞でC
a2+依存性エンドサイトーシスに関与し、「Rab3D」は脂
肪細胞でインスリン依存性のグルコーストランスポータ
ーの輸送に関与していることが示唆されている。従っ
て、Rab3サブファミリーに特異的な制御因子が単離され
れば、神経伝達物質の放出やホルモン分泌の制御機構の
解明やその異常による疾患の病態の解明や治療法の開発
などに利用でき産業上非常に有用である。Rab3サブファ
ミリーのうち、Rab3Aに作用する「GEP」及び「GAP」は
ラット脳から部分的には精製されているが(Burstein,E.
S.,et al.(1991) J.Biol.Chem.266,2689-2692、Burstei
n,E.S., and Macara,I.G.(1992) Proc.Natl.Acad.Sci.
U.S.A.89,1154-1158)、これら分子の一次構造及び正確
な特性（例えば、Rab3Aに対する特異性など）について
は明らかになっていない。即ち、Rab3サブファミリーに
特異的な制御因子についてはほとんど解明されていない
のが現状である。[0004] By the way, among the Rab subfamily, Rab
The three subfamilies include “Rab3A”, “Rab3B”, “Rab3C”,
And four members of “Rab3D” (Pfeff
er, SR (1994) Curr. Opin. Cell Biol. 6, 522-526). Among these members, “Rab3A” and “Rab3C” are involved in Ca 2+ -dependent exocytosis, and in particular, are involved in the vertebrate's critical function of neurotransmitter release from nerve endings. On the other hand, "Rab3B" is a pituitary cell
Involved in a 2+ -dependent endocytosis, suggesting that “Rab3D” is involved in insulin-dependent transport of glucose transporters in adipocytes. Therefore, if a regulatory factor specific to the Rab3 subfamily can be isolated, it can be used for elucidation of the mechanism of control of neurotransmitter release and hormone secretion, elucidation of disease pathology due to its abnormalities, and development of therapeutic methods. Above is very useful. Of the Rab3 subfamily, `` GEP '' and `` GAP '' that act on Rab3A have been partially purified from rat brain (Burstein, E.
S., et al. (1991) J. Biol. Chem. 266, 2689-2692, Burstei
n, ES, and Macara, IG (1992) Proc.Natl.Acad.Sci.
USA 89, 1154-1158), the primary structure and exact properties of these molecules (eg, specificity for Rab3A, etc.) are not known. That is, at present, little is known about regulatory factors specific to the Rab3 subfamily.
【０００５】[0005]
【発明が解決しようとする課題】本発明は、Rab3サブフ
ァミリーに特異的な制御因子を単離することを課題とす
る。An object of the present invention is to isolate a regulator specific to the Rab3 subfamily.
【０００６】[0006]
【課題を解決するための手段】本発明者らは上記課題を
解決すべく鋭意研究を行った結果、基質として脂質修飾
されたRab3Aを用いた連続的なカラムクロマトグラフィ
ーによりラットの脳のシナプス可溶性分画からGAPを精
製し単離することに成功した。さらに、本発明者らは、
単離したGAPの配列を基に作製したプローブを用いて、
該GAPをコードするヒトcDNAを単離することに成功し
た。本発明者らは、単離したGAPについてその機能を解
析したところ、該タンパク質が、脂質修飾されたRab3サ
ブファミリーメンバーに結合したGTPの加水分解を特異
的に促進する活性を有することを見いだした。Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that synaptic solubility in rat brain is determined by continuous column chromatography using lipid-modified Rab3A as a substrate. GAP was successfully purified and isolated from the fractions. In addition, we have:
Using a probe prepared based on the sequence of the isolated GAP,
The human cDNA encoding the GAP was successfully isolated. The present inventors have analyzed the function of the isolated GAP and found that the protein has an activity of specifically promoting the hydrolysis of GTP bound to a lipid-modified Rab3 subfamily member. .
【０００７】即ち、本発明はRab3サブファミリーメンバ
ーに特異的な制御因子に関し、より具体的には、（１）
脂質修飾されたRab3サブファミリーメンバーに結合し
たGTPの加水分解を特異的に促進する活性を有するタン
パク質、（２） 哺乳類由来である（１）に記載のタン
パク質、（３） 配列番号：１に記載のタンパク質、ま
たは該タンパク質中のアミノ酸配列において１若しくは
数個のアミノ酸が置換、欠失、若しくは付加したアミノ
酸配列を有し、脂質修飾されたRab3サブファミリーメン
バーに結合したGTPの加水分解を特異的に促進する活性
を有するタンパク質、（４） 配列番号：２に記載のDN
AとハイブリダイズするDNAがコードするタンパク質であ
って、脂質修飾されたRab3サブファミリーメンバーに結
合したGTPの加水分解を特異的に促進する活性を有する
タンパク質、（５） （１）〜（４）に記載のタンパク
質をコードするDNA、（６） （５）に記載のDNAを含む
ベクター、（７） （６）に記載のベクターを保持する
形質転換体、（８） （７）に記載の形質転換体により
産生される組み換えタンパク質、に関する。That is, the present invention relates to a regulator specific to a member of the Rab3 subfamily, and more specifically, (1)
A protein having an activity of specifically promoting the hydrolysis of GTP bound to a lipid-modified Rab3 subfamily member, (2) a protein according to (1), which is derived from a mammal, and (3) a SEQ ID NO: 1. Has the amino acid sequence in which one or several amino acids have been substituted, deleted or added in the amino acid sequence of the protein, and specifically hydrolyzes GTP bound to a lipid-modified Rab3 subfamily member. (4) the DN of SEQ ID NO: 2
A protein encoded by a DNA that hybridizes with A and having an activity of specifically promoting the hydrolysis of GTP bound to a lipid-modified Rab3 subfamily member, (5) (1) to (4) (6) a vector containing the DNA of (5); (7) a transformant carrying the vector of (6); (8) a transformant of (7); Recombinant proteins produced by the transformants.
【０００８】[0008]
【発明の実施の形態】本発明は、脂質修飾されたRab3サ
ブファミリーメンバーに結合したGTPの加水分解を特異
的に促進する活性を有するタンパク質に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a protein having an activity of specifically promoting the hydrolysis of GTP bound to a lipid-modified Rab3 subfamily member.
【０００９】本発明のタンパク質に含まれる配列番号：
１に記載のタンパク質は、基質として脂質修飾されたRa
b3Aを用いた連続的なカラムクロマトグラフィーにより
ラットの脳のシナプス可溶性分画から精製されたタンパ
ク質である。該タンパク質は、Rab3サブファミリーメン
バー(Rab3A, Rab3B,Rab3C, Rab3D)に特異的に作用す
る。また、脂質修飾を受けたRab3にのみ作用し、脂質修
飾を受けないRab3には作用しない。これらの事実から配
列番号：１に記載のタンパク質は、例えば、神経細胞で
は神経伝達物質の放出に関与した活性型Rab3サブファミ
リーメンバーに作用して、該メンバーを不活性型に変化
させ、神経伝達物質の放出を阻害するなどの機能を有し
ていると考えられる。[0009] SEQ ID NO: contained in the protein of the present invention:
The protein according to 1 is a lipid-modified Ra as a substrate.
It is a protein purified from a synaptic soluble fraction of rat brain by continuous column chromatography using b3A. The protein acts specifically on Rab3 subfamily members (Rab3A, Rab3B, Rab3C, Rab3D). In addition, it acts only on Rab3 that has undergone lipid modification, and does not act on Rab3 that has not undergone lipid modification. Based on these facts, the protein described in SEQ ID NO: 1 acts on, for example, an activated Rab3 subfamily member involved in the release of a neurotransmitter in a nerve cell to change the member to an inactive form, and It is considered to have functions such as inhibiting the release of a substance.
【００１０】なお、当業者にとっては、周知技術である
部位特異的変異誘発法（Sambruck,J.,Fritsch,E.F.,and
Maniatis,T.(1989) Molecular Cloning: A Laborator
y,Manual,Cold Spring Harbor Laboratory,Cold Spring
Harbor,NY）などを用いて、配列番号：１に記載のタン
パク質中のアミノ酸を適宜置換などすることにより配列
番号：１に記載のタンパク質の機能的同等物を得ること
は常套手段である。従って、配列番号：１に記載のタン
パク質中のアミノ酸配列において1もしくは数個のアミ
ノ酸が置換、欠失もしくは付加されたアミノ酸配列を有
し、脂質修飾されたRab3サブファミリーメンバーに結合
したGTPの加水分解を特異的に促進する活性を有するタ
ンパク質も本発明の範囲に含まれる。For those skilled in the art, a site-directed mutagenesis method (Sambruck, J., Fritsch, EF, and
Maniatis, T. (1989) Molecular Cloning: A Laborator
y, Manual, Cold Spring Harbor Laboratory, Cold Spring
It is a common practice to obtain a functional equivalent of the protein of SEQ ID NO: 1 by appropriately substituting amino acids in the protein of SEQ ID NO: 1 using, for example, Harbor, NY). Therefore, the amino acid sequence of the protein described in SEQ ID NO: 1 has an amino acid sequence in which one or several amino acids have been substituted, deleted or added, and the GTP hydrolyzed to a lipid-modified Rab3 subfamily member. A protein having an activity of specifically promoting degradation is also included in the scope of the present invention.
【００１１】また、当業者にとっては、周知技術である
ハイブリダイゼーション技術（Sambruck,J.,Fritsch,E.
F.,and Maniatis,T.(1989) Molecular Cloning: A Labo
ratory,Manual,Cold Spring Harbor Laboratory,Cold S
pring Harbor,NY）を用いて、配列番号：２に記載のDNA
配列（またはその一部）を基に、これと相同性の高いDN
Aを単離して、該DNAから本発明のタンパク質の機能的同
等物を得ることも常套手段である。従って、配列番号：
２に記載のDNA配列からなるDNAとハイブリダイズするDN
Aがコードするタンパク質であって、脂質修飾されたRab
3サブファミリーメンバーに結合したGTPの加水分解を特
異的に促進する活性を有するタンパク質も本発明の範囲
に含まれる。ハイブリダイズ技術により得られたタンパ
ク質は、本発明のタンパク質とアミノ酸配列において70
％以上の相同性を有することが好ましく、80％以上の相
同性を有することがさらに好ましく、90％以上の相同性
を有することがさらに好ましい。[0011] Also, those skilled in the art will recognize hybridization techniques (Sambruck, J., Fritsch, E.
F., and Maniatis, T. (1989) Molecular Cloning: A Labo
ratory, Manual, Cold Spring Harbor Laboratory, Cold S
The DNA of SEQ ID NO: 2 was obtained by using Spring Harbor, NY).
Based on the sequence (or part of it), a DN with high homology to this
It is also conventional to isolate A and obtain a functional equivalent of the protein of the invention from the DNA. Thus, SEQ ID NO:
2. A DN that hybridizes with the DNA consisting of the DNA sequence described in 2.
A is a protein encoded by a lipid-modified Rab
Proteins having the activity of specifically promoting the hydrolysis of GTP bound to the three subfamily members are also included in the scope of the present invention. The protein obtained by the hybridization technique is the same as the protein of the present invention in amino acid sequence.
% Or more, more preferably 80% or more, and even more preferably 90% or more.
【００１２】なお、本発明において、「脂質修飾された
Rab3サブファミリーメンバーに結合したGTPの加水分解
を特異的に促進する活性」とは、脂質修飾されたRab3サ
ブファミリーメンバーに結合したGTPの加水分解を促進
し、脂質修飾されていないRab3サブファミリーメンバー
に結合したGTP、およびRab3以外のサブファミリーメン
バーに結合したGTPの加水分解を実質的に促進しない活
性を指す。脂質修飾されたRab3サブファミリーメンバー
に結合したGTPの加水分解を特異的に促進する活性は、
後述するRab3GAPの標準的測定、overlay法などの方法で
検出することが可能である。In the present invention, "lipid-modified
`` Activity that specifically promotes hydrolysis of GTP bound to Rab3 subfamily members '' refers to Rab3 subfamily members that promote hydrolysis of GTP bound to lipid-modified Rab3 subfamily members and are not lipid-modified. Refers to an activity that does not substantially promote hydrolysis of GTP bound to GTP and GTP bound to subfamily members other than Rab3. The activity of specifically promoting the hydrolysis of GTP bound to a lipid-modified Rab3 subfamily member is as follows:
It can be detected by standard measurement of Rab3GAP described later, an overlay method, or the like.
【００１３】本発明のタンパク質は、後述するカラムク
ロマトグラフィーなどにより調製できる。また、本発明
のタンパク質は、組み換えタンパク質として調製するこ
とも可能である。組み換えタンパク質は、例えば、本発
明のタンパク質をコードするDNA（例えば、配列番号：
２に記載のDNA）を適当なベクターに組み込んで宿主細
胞に導入し、得られた形質転換体内で発現したタンパク
質を精製することにより調製することが可能である。The protein of the present invention can be prepared by column chromatography described below. Further, the protein of the present invention can be prepared as a recombinant protein. The recombinant protein is, for example, a DNA encoding the protein of the present invention (for example, SEQ ID NO:
2) can be prepared by incorporating the DNA into an appropriate vector, introducing it into host cells, and purifying the protein expressed in the resulting transformant.
【００１４】また、本発明は、上記本発明のタンパク質
をコードするDNAに関する。本発明のDNAの形態には、特
に制限はない。mRNAから合成されたcDNA、ゲノムDNAの
他、化学合成DNAなども本発明のDNAに含まれる。The present invention also relates to a DNA encoding the protein of the present invention. The form of the DNA of the present invention is not particularly limited. In addition to cDNA and genomic DNA synthesized from mRNA, chemically synthesized DNA and the like are also included in the DNA of the present invention.
【００１５】本発明のDNAは常法（例えば、cDNAであれ
ば、逆転写酵素等を用いてmRNAから合成後、アルカリSD
S法および塩化セシウム密度勾配遠心法などにより調製
する）により調製することが可能である。The DNA of the present invention can be prepared by a conventional method (for example, in the case of cDNA, after synthesis from mRNA using reverse transcriptase, etc.
Prepared by the method S and the cesium chloride density gradient centrifugation method).
【００１６】また、本発明は、本発明のDNAが挿入され
たベクターに関する。本発明のDNAが挿入されるベクタ
ーとしては特に制限はなく、例えば、クローニング用ベ
クターであれば、「pBlueScript」、「pGEM」などが挙
げられ、また哺乳動物細胞発現用ベクターであれば、
「pEF-BOS」、「pSRα」、「pCMV」などが挙げられる。[0016] The present invention also relates to a vector into which the DNA of the present invention has been inserted. The vector into which the DNA of the present invention is inserted is not particularly limited.For example, a cloning vector includes `` pBlueScript '', `` pGEM '', and a mammalian cell expression vector.
"PEF-BOS", "pSRα", "pCMV" and the like.
【００１７】本発明のタンパク質を生産する目的におい
てベクターを使用する場合には、特に発現ベクターが有
用である。発現ベクターとしては、例えば、大腸菌用で
あれば、「pGEXZT」、「pRSET」、「pTvCHis」などが挙
げられ、SF9細胞用であれば、「pACYM1」などが挙げら
れ、哺乳動物細胞用では、「pEF-Bos」、「pSRα」、
「pCMV」、「pBlueScript」等が挙げられるが、これら
に制限されない。When a vector is used for producing the protein of the present invention, an expression vector is particularly useful. Examples of expression vectors include, for Escherichia coli, `` pGEXZT '', `` pRSET '', `` pTvCHis '', and the like.For SF9 cells, `` pACYM1 '' and the like.For mammalian cells, “PEF-Bos”, “pSRα”,
Examples include, but are not limited to, "pCMV", "pBlueScript", and the like.
【００１８】ベクターへの本発明のDNAの挿入は、例え
ば、文献（Sambruck,J.,Fritsch,E.F.,and Maniatis,T.
(1989) Molecular Cloning: A Laboratory,Manual,Cold
Spring Harbor Laboratory,Cold Spring Harbor,NY）
に記載の方法により行うことができる。Insertion of the DNA of the present invention into a vector is described, for example, in the literature (Sambruck, J., Fritsch, EF, and Maniatis, T .;
(1989) Molecular Cloning: A Laboratory, Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY)
Can be performed.
【００１９】また、本発明は、本発明のベクターが導入
された宿主細胞に関する。本発明のベクターが導入され
る宿主細胞としては、本発明のベクターに適合する細胞
であれば特に制限はなく、種々の動物細胞（例えば、天
然の細胞の他、COS細胞、PC12細胞などの株化された細
胞など）、細菌、酵母、昆虫細胞などが挙げられる。本
発明のタンパク質の製造目的であれば、特に、大腸菌、
SF9細胞などが好適である。The present invention also relates to a host cell into which the vector of the present invention has been introduced. The host cell into which the vector of the present invention is introduced is not particularly limited as long as it is a cell compatible with the vector of the present invention, and various animal cells (eg, natural cells, strains such as COS cells, PC12 cells, etc.) Cells, etc.), bacteria, yeast, insect cells and the like. For the purpose of producing the protein of the present invention, in particular, E. coli,
SF9 cells and the like are preferred.
【００２０】宿主細胞へのベクターの導入は、例えば、
文献（Sambruck,J.,Fritsch,E.F.,and Maniatis,T.(198
9) Molecular Cloning: A Laboratory,Manual,Cold Spr
ingHarbor Laboratory,Cold Spring Harbor,NY）記載の
方法に従って行うことが可能である。The introduction of a vector into a host cell can be performed, for example, by
References (Sambruck, J., Fritsch, EF, and Maniatis, T. (198
9) Molecular Cloning: A Laboratory, Manual, Cold Spr
ingHarbor Laboratory, Cold Spring Harbor, NY).
【００２１】形質転換体内で発現した本発明の組み換え
タンパク質は、種々のクロマトグラフィー、電気泳動
法、ゲル濾過などの常法を適宜組み合わせて精製するこ
とが可能である。また、例えば、本発明のタンパク質を
GSTやHisbとの融合タンパク質として発現させる場合に
は、それぞれグルタチオンセファロースカラム、ニッケ
ルセファロースカラムを用いて精製することも可能であ
る。The recombinant protein of the present invention expressed in a transformant can be purified by appropriately combining various conventional methods such as chromatography, electrophoresis and gel filtration. Also, for example, the protein of the present invention
When expressed as a fusion protein with GST or Hisb, purification can be performed using a glutathione sepharose column and a nickel sepharose column, respectively.
【００２２】以下、本発明を実施例により具体的に説明
するが、本発明はこれら実施例に制限されるものではな
い。Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.
【００２３】[0023]
[実施例１] 試料及び化学物質 脂質修飾及び非修飾のRab3Aは、Rab3A cDNAを持つバキ
ュロウイルス(baculovirus)で感染させたSf(Spodoptera
frugiperda)9細胞の膜及び可溶性分画それぞれから精
製した(Kikuchi,A.,et al.(1995) Methods Enzymol.25
7,57-70)。Rab2、Rab3B、Rab3C、Rab3D、Rab5A、及びRa
b11の脂質修飾体を、各cDNAを持つバキュロウイルスで
感染させたSf9細胞の膜分画から同様の方法で精製し
た。なお、[γ-3 2P]GTP(185 TBq/mmol)及び、[α-32P]G
TP(110 TBq/mmol)はアマーシャム社より購入した。[Example 1] Samples and chemical substances Lipid-modified and unmodified Rab3A were infected with a baculovirus carrying a Rab3A cDNA, Sf (Spodoptera).
frugiperda) Purified from the membrane and soluble fraction of 9 cells, respectively (Kikuchi, A., et al. (1995) Methods Enzymol. 25)
7,57-70). Rab2, Rab3B, Rab3C, Rab3D, Rab5A, and Ra
The modified lipid of b11 was purified in the same manner from the membrane fraction of Sf9 cells infected with baculovirus having each cDNA. Incidentally, [γ- 3 2 P] GTP (185 TBq / mmol) and, [α- 32 P] G
TP (110 TBq / mmol) was purchased from Amersham.
【００２４】[実施例２] Rab3 GAPの標準的測定 脂質修飾されたRab3A(3pmol)を、「25mM Tris/Hcl(pH8.
0)、10mM EDTA、5mM MgCl2、0.5mM DTT、0.3% CHAPS、
及び1.5μM[γ-32P]GTP(1×104 cpm/pmol)」を含む反応
混合液(10μl)中、30℃で10分間インキュベートした。
この反応を2.5μlの80mM MgCl2を加えて停止した。この
混合液(12.5μl)に、測定するサンプルを総量50μlにな
るように加え、さらに30 ℃で5分間インキュベートし
た。この混合液をニトロセルロースフィルターに流し、
フィルター上の残留放射活性をチェレンコフカウンティ
ング(Cerenkov counting)により決定した。Example 2 Standard Measurement of Rab3 GAP Lipid-modified Rab3A (3 pmol) was added to 25 mM Tris / Hcl (pH 8.
0), 10 mM EDTA, 5 mM MgCl 2 , 0.5 mM DTT, 0.3% CHAPS,
And 1.5 μM [γ- 32 P] GTP (1 × 10 4 cpm / pmol) ”in a reaction mixture (10 μl) at 30 ° C. for 10 minutes.
The reaction was stopped by adding 2.5 μl of 80 mM MgCl 2 . The sample to be measured was added to this mixture (12.5 μl) so that the total amount of the sample became 50 μl, and the mixture was further incubated at 30 ° C. for 5 minutes. Pour this mixture through a nitrocellulose filter,
Residual radioactivity on the filters was determined by Cerenkov counting.
【００２５】[実施例３] 「overlay法」によるRab3GAP
活性の測定 Rab3GAPの精製したサンプルに対しポリアクリルアミド
ゲル電気泳動(SDS-PAGE)を行った（図2A）。セミドライ
(semi-dry)ウエスタンブロッティング後、ニトロセルロ
ースフィルターに結合したタンパク質を、「1％ウシ血
清アルブミン、0.5mM MgCl2、0.1％Triton X-100、及び
5mM DTT」を含むリン酸緩衝生理食塩水中で処理し、変
性を回復させた。Rab3GAP活性を検知するため、フィル
ターを「25mM HEPES/NaOH(pH7.0)、0.05％ Triton X-10
0、1.25mM MgCl2、及び2.5mM DTT」を含む緩衝液中で
[γ-32P]GTP-Rab3Aとともに25℃で10分間インキュベー
トした。フィルターを、「25mM HEPES/NaOH(pH7.0)、5m
M MgCl2、及び2.5mM DTT」を含むリン酸緩衝生理食塩水
中で洗浄し、放射活性を「Fujix BAS 2000 Imaging Ana
lyzer」で測定した。この結果、分子量約130kDaの位置
にRab3GAPのバンドが検出された（図には示していな
い）。[Example 3] Rab3GAP by "overlay method"
Measurement of activity The purified sample of Rab3GAP was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 2A). Semi-dry
(Semi-dry) After Western blotting, the protein bound to the nitrocellulose filter was separated into 1% bovine serum albumin, 0.5 mM MgCl 2 , 0.1% Triton X-100, and
Treatment was performed in phosphate buffered saline containing "5 mM DTT" to restore denaturation. In order to detect Rab3GAP activity, filter was changed to 25 mM HEPES / NaOH (pH 7.0), 0.05% Triton X-10.
0,1.25mM MgCl 2, and 2.5 mM DTT "in buffer containing
Incubated with [γ- 32 P] GTP-Rab3A at 25 ° C for 10 minutes. Filter the filter using `` 25 mM HEPES / NaOH (pH 7.0),
Wash with phosphate buffered saline containing M MgCl 2 and 2.5 mM DTT, and measure the radioactivity on the Fujix BAS 2000 Imaging Ana
lyzer ". As a result, a Rab3GAP band was detected at a position having a molecular weight of about 130 kDa (not shown in the figure).
【００２６】[実施例４] Rab3GAPの精製 すべての精製は0〜4℃で行った。シナプス可溶性(SS)分
画を200匹のラット脳から調製し(Mizoguchi,A.,et al.
(1990) J.Biol.Chem.265,11872-11879)、その5分の1のS
S分画(450ml、315mg)を「緩衝液A」（20mM Tris/Hcl(pH
7.5)、0.5mM EDTA、及び1mM DTT)で平衡したQ-セファロ
ースFFカラム(2.6×23cm)を用いて分画した。600mlの
「緩衝液A」でカラムを洗浄後、溶出を600mlの「緩衝液
A」中のNaClの直線勾配(0-0.5M)、続いて120mlの「緩衝
液A」中の0.5M NaClを用いて流出速度5ml/分で行い、8m
lずつの分画を集めた。この結果、Rab3GAP活性の１つの
ピークが分画62-70で検出された。次いで、これらの分
画(72ml、36mg)を集め、一方、残りのSS分画について同
様の方法で同じQ-セファロースFFカラムクロマトグラフ
ィーを4回行った。 5回のQ-セファロースFFカラムクロ
マトグラフィーのサンプルをプールし、「緩衝液B」（2
0mM リン酸カリウム(pH7.5)、及び1mM DTT)720mlで希釈
した。そのサンプルを「緩衝液B」で平衡させたヒドロ
キシアパタイトカラム(2.6×6.6cm)を用いてさらに分画
した。同じ緩衝液350mlでカラムを洗浄後、溶出を「緩
衝液B」中の500mlのリン酸カリウムの直線勾配(20-212m
M)、続いて「緩衝液B」中の150mlの直線勾配(212-500m
M)及び150mlの500mM リン酸カリウムを用いて流出速度
1.25ml/分で行い、10mlずつの分画を集めた。この結
果、Rab3GAP活性の１つのピークが分画29-40で検出され
た。次いで、この分画(120ml、18mg)を集め、240mlの
「緩衝液A」で希釈した。そのサンプルを「緩衝液A」で
平衡させたヘパリン-セファロースCL-6Bカラム(0.5×5c
m)を用いてさらに分画した。同じ緩衝液20mlでカラムを
洗浄後、溶出を「緩衝液A」中の0.5M NaClを用いて流出
速度0.5ml/分で行い、1mlずつの分画を集めた。この結
果、Rab3GAP活性の１つのピークが分画2-6で見られた。
これらの分画(5ml、4mg)を集め、その5分の1を2mlの
「緩衝液A」で希釈して「緩衝液A」中で2mlの280mM NaC
lで平衡させたモノQ PC 1.6/5カラムを用いて分画し
た。2mlの同じ緩衝液でカラムを洗浄後、溶出を、「緩
衝液A」中の3mlのNaClの直線勾配(280-500mM)、続いて
0.5mlのNaClの直線勾配及び緩衝液A中の0.5mlの1M NaCl
を用いて流出速度0.1ml/分で行い、0.1mlずつの分画を
集めた。この結果、Rab3GAP活性の１つのピークが分画1
0及び11で検出された(図1参照)。これらの分画(0.2ml、
14μg)を集め、一方、残りのヘパリン-セファロースの
サンプルについては、同様の方法でモノQ カラムクロマ
トグラフィーを4回行った。5回のモノQ カラムクロマト
グラフィーのサンプルをプールして、使用時まで-80℃
で保存した。Example 4 Purification of Rab3GAP All purifications were performed at 0-4 ° C. Synaptic soluble (SS) fractions were prepared from 200 rat brains (Mizoguchi, A., et al.
(1990) J. Biol. Chem. 265, 11872-11879), one fifth of which S
The S fraction (450 ml, 315 mg) was added to "Buffer A" (20 mM Tris / Hcl (pH
The fractionation was performed using a Q-Sepharose FF column (2.6 × 23 cm) equilibrated with 7.5), 0.5 mM EDTA, and 1 mM DTT). After washing the column with 600 ml of buffer A, elute
A linear gradient (0-0.5 M) of NaCl in `` A '', followed by an efflux rate of 5 ml / min using 120 ml of 0.5 M NaCl in `` buffer A '', 8 m
Fractions of l were collected. As a result, one peak of Rab3GAP activity was detected in fractions 62-70. These fractions (72 ml, 36 mg) were then collected, while the remaining SS fractions were subjected to the same Q-Sepharose FF column chromatography four times in a similar manner. Pool the samples from the five Q-Sepharose FF column chromatography columns and add “Buffer B” (2
It was diluted with 720 ml of 0 mM potassium phosphate (pH 7.5) and 1 mM DTT. The sample was further fractionated using a hydroxyapatite column (2.6 × 6.6 cm) equilibrated with “Buffer B”. After washing the column with 350 ml of the same buffer, the elution was performed with a linear gradient (20-212 m) of 500 ml of potassium phosphate in "Buffer B".
M) followed by a linear gradient of 150 ml in `` buffer B '' (212-500 m
M) and efflux rate using 150 ml of 500 mM potassium phosphate
Performed at 1.25 ml / min, collecting 10 ml fractions. As a result, one peak of Rab3GAP activity was detected in fraction 29-40. The fractions (120 ml, 18 mg) were then collected and diluted with 240 ml of "Buffer A". A heparin-Sepharose CL-6B column (0.5 × 5 c
Further fractionation was performed using m). After washing the column with 20 ml of the same buffer, elution was carried out with 0.5 M NaCl in "Buffer A" at an effluent rate of 0.5 ml / min, and 1 ml fractions were collected. As a result, one peak of Rab3GAP activity was observed in fraction 2-6.
These fractions (5 ml, 4 mg) were collected, one fifth was diluted with 2 ml of `` buffer A '' and 2 ml of 280 mM NaC in `` buffer A ''
Fractionation was performed using a Mono Q PC 1.6 / 5 column equilibrated in l. After washing the column with 2 ml of the same buffer, elution was performed with a linear gradient (280-500 mM) of 3 ml of NaCl in `` buffer A '', followed by
0.5 ml NaCl linear gradient and 0.5 ml 1 M NaCl in buffer A
Was carried out at a flow rate of 0.1 ml / min, and fractions of 0.1 ml each were collected. As a result, one peak of Rab3GAP activity was identified as fraction 1
Detected at 0 and 11 (see FIG. 1). These fractions (0.2 ml,
14 μg) was collected, while the remaining Heparin-Sepharose sample was subjected to Mono Q column chromatography four times in the same manner. Pool samples from 5 Mono Q column chromatography and store at -80 ° C until use
Saved in.
【００２７】最後のモノQ カラムクロマトグラフィーに
おいて、GAP活性は約150kDa及び約130kDaのMrを持つ２
つのタンパク質の溶出パターンと一致した(図1A及び
B)。このサンプルをスクロース密度勾配超遠心分離(ult
racentrifugation)で分画し、さらにモノQ カラムクロ
マトグラフィーを行ったところ、GAP活性はこの2つのタ
ンパク質の溶出パターンと一致した(図１C及びD)。スク
ロース密度勾配超遠心分離によって評価されたMrは約29
0kDaだった。これらの結果からRab3GAPは150kDa及び130
kDaのタンパク質のヘテロダイマーであると推定され
る。In the last Mono Q column chromatography, the GAP activity was 2% with Mr of about 150 kDa and about 130 kDa.
(Fig.1A and
B). The sample is centrifuged by sucrose density gradient ultracentrifugation (ult
GAP activity was consistent with the elution patterns of the two proteins when fractionated by racentrifugation) and further subjected to mono Q column chromatography (FIGS. 1C and D). Mr estimated by sucrose density gradient ultracentrifugation is approximately 29
It was 0kDa. From these results, Rab3GAP was 150 kDa and 130 kDa.
Presumed to be a heterodimer of kDa protein.
【００２８】[実施例５] 薄層クロマトグラフィーによ
るRab3GAP活性の測定 [α-32P]GTP-Rab3Aを、[γ-32P]GTPの代わりに[α-32P]
GTP用いたことを除いて実施例２の方法と同様に調製
し、ニトロセルロースフィルターに流した。フィルター
を洗浄後、「20mM Tris/Hcl(pH8.0)、20mM EDTA、2％ S
DS、1mM GDP、及び1mM GTP」を含む緩衝液に65℃で5分
間、フィルターを浸すことによりRab3Aに結合したグア
ニンヌクレオチドを溶出した。放出されたヌクレオチド
を、展開液としての1M リン酸カリウムを用いてポリエ
チレンイミン-セルロース(マシェレー-ネーゲル(Masher
ey-Nagel)社製)クロマトグラフィーにより分離し、放射
活性を、「Fujix BAS 2000 Imaging Analyzer」で測定
した。Example 5 Measurement of Rab3GAP Activity by Thin-Layer Chromatography [α- 32 P] GTP-Rab3A was replaced by [α- 32 P] instead of [γ- 32 P] GTP.
Except that GTP was used, it was prepared in the same manner as in Example 2 and passed through a nitrocellulose filter. After washing the filter, “20mM Tris / Hcl (pH8.0), 20mM EDTA, 2% S
Guanine nucleotides bound to Rab3A were eluted by immersing the filter in a buffer containing DS, 1 mM GDP, and 1 mM GTP at 65 ° C. for 5 minutes. The released nucleotides were subjected to polyethyleneimine-cellulose (Masher-Nagel) using 1 M potassium phosphate as a developing solution.
ey-Nagel), and the radioactivity was measured with a “Fujix BAS 2000 Imaging Analyzer”.
【００２９】この結果、最初のモノQ カラムクロマトグ
ラフィーからのRab3GAPサンプルに薄層クロマトグラフ
ィーによって評価されたGAP活性が確かに示された(図2
B)。脂質修飾Rab3Aにおいては活性を持つが、非修飾体
では活性は持たなかった(図3A)。多くのRabファミリー
において、Rab3A、Rab3B、Rab3C、及びRab3Dを含むRab3
サブファミリーにRab3GAPは活性を示し、Rab2、Rab5A、
及びRab11を含む他のRabサブファミリーには活性を示さ
なかった(図3B)。Rab3サブファミリーにおいて、Rab3GA
PはRab3A、Rab3C、及びRab3Dには強い活性を示したが、
Rab3Bには弱い活性しか示さなかった。The results clearly demonstrated that the Rab3GAP sample from the first Mono Q column chromatography exhibited GAP activity as assessed by thin layer chromatography (FIG. 2).
B). It was active in the lipid-modified Rab3A, but not in the unmodified form (FIG. 3A). In many Rab families, Rab3 including Rab3A, Rab3B, Rab3C, and Rab3D
Rab3GAP shows activity in subfamilies, Rab2, Rab5A,
And other Rab subfamilies, including Rab11, showed no activity (FIG. 3B). In the Rab3 subfamily, Rab3GA
P showed strong activity on Rab3A, Rab3C, and Rab3D,
Rab3B showed only weak activity.
【００３０】[実施例６] アミノ酸配列の測定 200匹のラット脳から一連のカラムクロマトグラフィー
によって集めたRab3GAPのモノQサンプル(13μg)に対しS
DS-PAGEを行った。Rab3GAPに対応するタンパク質（130k
Daのタンパク質）バンドをゲルから切り取り、リシルエ
ンドペプチダーゼ(lysyl endopeptidase)で切断した。
切断されたペプチドは、C18逆相高圧液体カラムクロマ
トグラフィーにより分離した(Imazumi,K.,et al.(1994)
Biochem.Biophys.Res.Commun.205,1409-1416)。このペ
プチドのいくつかのアミノ酸配列をペプチドシーケンサ
ー(Shimazu PSQ-1-gas phase sequencer)で決定した。Example 6 Determination of Amino Acid Sequence A mono-Q sample (13 μg) of Rab3GAP collected from a series of 200 rat brains by a series of column chromatography was analyzed using S
DS-PAGE was performed. Protein corresponding to Rab3GAP (130k
The Da protein) band was excised from the gel and cut with lysyl endopeptidase.
The cleaved peptide was separated by C18 reverse-phase high-pressure liquid column chromatography (Imazumi, K., et al. (1994).
Biochem. Biophys. Res. Commun. 205, 1409-1416). Some amino acid sequences of this peptide were determined with a peptide sequencer (Shimazu PSQ-1-gas phase sequencer).
【００３１】この結果、30以上のペプチドピークが見ら
れ、3つのアミノ酸配列が決定された。これらの配列
は、「PVPARRQRRLFDDTREAEK」（配列番号：３）、「LTE
PAPVPIHK」（配列番号：４）、及び「DMAPLKPEGRLHQHG
K」（配列番号：５）であった。さらに、130kDaのタン
パク質のN末端アミノ酸配列を決定するためにモノQサン
プルの一部をSDS-PAGEにかけ、次にPVDF膜に転写したタ
ンパク質をクーマジーブリリアントブルー(Coomassie b
rilliant blue)で染色した。このタンパク質に対応する
タンパク質バンドを膜から切り離してペプチドシーケン
サーに直接かけた。この結果、130kDaのタンパク質のN
末端アミノ酸の配列が決定され、「AADSEPESEV」（配列
番号：６）であると判明した。As a result, 30 or more peptide peaks were observed, and three amino acid sequences were determined. These sequences are “PVPARRQRRLFDDTREAEK” (SEQ ID NO: 3), “LTE
PAPVPIHK "(SEQ ID NO: 4) and" DMAPLKPEGRLHQHG
K "(SEQ ID NO: 5). In addition, a portion of the Mono-Q sample was subjected to SDS-PAGE to determine the N-terminal amino acid sequence of the 130 kDa protein, and the protein transferred to the PVDF membrane was then transferred to Coomassie brilliant blue (Coomassie b).
rilliant blue). The protein band corresponding to this protein was cut off the membrane and applied directly to a peptide sequencer. This results in a 130 kDa protein N
The sequence of the terminal amino acid was determined and found to be "AADSEPESEV" (SEQ ID NO: 6).
【００３２】[実施例７] Rab3GAPの核酸配列の分子ク
ローニング及び決定 ヒト脳cDNAライブラリーのスクリーニングにおけるハイ
ブリダイゼーションの方法は常法(Sambrook,J.,et al.
(1989) Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold Spring Harbor Laboratory, Cold Spring H
arbor, NY)にて行った。λgt10ファージベクターで得ら
れるcDNAクローンをpBluescriptプラスミドを用いて再
びクローン化した。Example 7 Molecular Cloning and Determination of Rab3GAP Nucleic Acid Sequence The hybridization method for screening a human brain cDNA library is a conventional method (Sambrook, J., et al.
(1989) Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold Spring Harbor Laboratory, Cold Spring H
arbor, NY). The cDNA clone obtained with the λgt10 phage vector was cloned again using the pBluescript plasmid.
【００３３】本発明者らはヒト脳cDNAライブラリーから
cDNAをクローン化し、一次構造を決定した。この結果、
ヒトcDNAは分子量が110521で981のアミノ酸を持つタン
パク質をコードしていることが判明した(図4)。決定し
た塩基配列を配列番号：２に、アミノ酸配列を配列番
号：１に示す。6個のアミノ酸を除いてペプチドのすべ
てのアミノ酸配列は配列番号：６の配列を含んでいた。
このアミノ酸配列のわずかな違いは種(species)の違い
のためであると考えられる。一方、コンピューターホモ
ロジーサーチ(computer homology search)により、この
配列がジーンバンクヌクレオチドシーケンスデータベー
ス(GenBank Nucleotide Sequence Database)にある機能
が未知であるアクセッション(accession)番号D31886の
遺伝子と同一であることが判明した。Rab3GAPをコード
するcDNAは既知のタンパク質とは有意な相同性は見出さ
れなかった。The present inventors have determined from a human brain cDNA library
The cDNA was cloned and the primary structure was determined. As a result,
The human cDNA was found to encode a protein having a molecular weight of 110521 and 981 amino acids (FIG. 4). The determined nucleotide sequence is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 1. All amino acid sequences of the peptide except for 6 amino acids included the sequence of SEQ ID NO: 6.
This slight difference in amino acid sequence is believed to be due to differences in species. On the other hand, a computer homology search revealed that this sequence was identical to the gene with accession number D31886 whose function in the GeneBank Nucleotide Sequence Database was unknown. . CDNA encoding Rab3GAP did not show significant homology to known proteins.
【００３４】[実施例８] 組換えRab3GAPの発現 発現プラスミド「pRSET-Rab3GAP」及び「pGEX-2T-Rab3
欠失変異体」の構築を以下の方法により行った。開始メ
チオニンコドンの上流及び終止コドンの下流にKpnI部位
の領域を付加したRab3GAP cDNAを含む2946塩基対断片を
PCR反応により合成した。この断片をKpnIで切断し、プ
ラスミド「pRSET」（Invitrogen社製）のKpnI部位へ挿
入した（得られたプラスミドを「pRSET-Rab3GAP」と命
名した）。「pRSET-Rab3GAP」で大腸菌DE3を形質転換し
た。30℃で4時間、1mM イソプロピル-β-D-チオガラク
トピラノシドを用いて発現を誘導後、細胞を50mMのリン
酸ナトリウム（pH7.4）及び50mM NaClを含む緩衝液中に
懸濁した。この懸濁液を超音波破砕し、遠心分離した。
この上清から、Hisb融合Rab3GAPをNi2+-NTA-アガロース
カラムクロマトグラフィーにより精製した。この融合タ
ンパク質を「50mMリン酸ナトリウム(pH7.4)、50mM NaC
l、及び0.5M イミダゾール」を含む緩衝液で溶出した。
欠失変異cDNA(1-909塩基対、910-1800塩基対、及び1801
-2946塩基対)をPCR反応により合成し、「pGEX-2T」（Ph
armacia社製）に挿入した（得られたプラスミドを「pGE
X-2T-Rab3GAP欠失変異体」と命名した）。「pGEX-2T-Ra
b3GAP欠失変異体」を大腸菌DH5αへ形質転換した。30℃
で30分、イソプロピル-β-D-チオガラクトピラノシドで
発現を誘導した後、細胞を10％スクロースを含む「緩衝
液A」中に懸濁した。この懸濁液を超音波破砕し、遠心
分離した。この上清から、GST融合Rab3GAP欠失変異体を
グルタチオン結合セファロースビーズにより精製した。Example 8 Expression of Recombinant Rab3GAP The expression plasmids “pRSET-Rab3GAP” and “pGEX-2T-Rab3”
The construction of "deletion mutant" was carried out by the following method. A 2946 base pair fragment containing a Rab3GAP cDNA with a KpnI site added upstream of the initiation methionine codon and downstream of the stop codon.
It was synthesized by PCR. This fragment was digested with KpnI and inserted into the KpnI site of plasmid "pRSET" (manufactured by Invitrogen) (the obtained plasmid was named "pRSET-Rab3GAP"). Escherichia coli DE3 was transformed with “pRSET-Rab3GAP”. After induction of expression with 1 mM isopropyl-β-D-thiogalactopyranoside at 30 ° C. for 4 hours, the cells were suspended in a buffer containing 50 mM sodium phosphate (pH 7.4) and 50 mM NaCl. . This suspension was sonicated and centrifuged.
From this supernatant, Hisb-fused Rab3GAP was purified by Ni 2+ -NTA-agarose column chromatography. This fusion protein was added to 50 mM sodium phosphate (pH 7.4), 50 mM NaC
l, and 0.5 M imidazole ".
Deletion mutant cDNA (1-909 base pairs, 910-1800 base pairs, and 1801
-2946 base pairs) was synthesized by PCR, and “pGEX-2T” (Ph
armacia) (the resulting plasmid was called "pGE
X-2T-Rab3GAP deletion mutant "). `` PGEX-2T-Ra
The “b3GAP deletion mutant” was transformed into E. coli DH5α. 30 ℃
After induction with isopropyl-β-D-thiogalactopyranoside for 30 minutes, the cells were suspended in “buffer A” containing 10% sucrose. This suspension was sonicated and centrifuged. From this supernatant, a GST-fused Rab3GAP deletion mutant was purified using glutathione-conjugated Sepharose beads.
【００３５】これにより130kDaの組み換えタンパク質が
His6融合タンパク質として調製された。この組換えタン
パク質はRab3Aに対しGAP活性を確かに示した(図3C)。ま
た他のRab3サブファミリーにも活性を示したが、他のRa
bファミリーには活性を示さなかった。配列番号：１の1
〜303番目のアミノ酸、304〜600番目のアミノ酸、及び6
01〜981アミノ酸残基の組換えタンパク質を用いた触媒
ドメイン解析により、少なくとも601〜981番目のアミノ
酸残基に触媒ドメイン存在することが示された。なお、
ノーザンブロット解析により、脳、心臓、骨格筋、腎
臓、肝臓、脾臓、膵臓、精巣、及び卵巣を含むすべての
ラットの組織において130kDaのタンパク質のmRNAが発現
することが判明した。As a result, a 130 kDa recombinant protein is obtained.
Prepared as His6 fusion protein. This recombinant protein did show GAP activity against Rab3A (FIG. 3C). It also showed activity on other Rab3 subfamilies, but other Rab
No activity was shown for the b family. SEQ ID NO: 1
Amino acids 303 to 303, amino acids 304 to 600, and 6
Catalytic domain analysis using a recombinant protein of 01 to 981 amino acid residues indicated that the catalytic domain was present at least at amino acids 601 to 981. In addition,
Northern blot analysis revealed that the mRNA for the 130 kDa protein was expressed in all rat tissues including brain, heart, skeletal muscle, kidney, liver, spleen, pancreas, testis, and ovary.
【００３６】[0036]
【発明の効果】本発明により、Rab3サブファミリーメン
バーに特異的に作用するタンパク質が提供された。本発
明のタンパク質は、特に神経伝達物質の放出に関与して
いると考えられるRab3サブファミリーメンバーの脂質結
合形態に高い特異性を示し、このメンバーに結合したGT
Pの加水分解を特異的に促進する活性を示した。本発明
の制御因子は、例えば、神経伝達物質の放出やホルモン
分泌の制御機構や疾患との関連の解明への利用が期待さ
れる。Industrial Applicability According to the present invention, a protein that specifically acts on a Rab3 subfamily member is provided. The proteins of the present invention show high specificity, especially for the lipid-bound form of the Rab3 subfamily members thought to be involved in the release of neurotransmitters,
It showed an activity to specifically promote the hydrolysis of P. The control factor of the present invention is expected to be used for elucidation of, for example, the mechanism of controlling neurotransmitter release and hormone secretion and its relationship to diseases.
【００３７】[0037]
配列番号 : 1 配列の長さ : 981 配列の型 : アミノ酸 トポロジー : 直鎖状 配列の種類 : タンパク質 配 列 Met Ala Ala Asp Ser Glu Pro Glu Ser Glu Val Phe Glu Ile Thr Asp 1 5 10 15 Phe Thr Thr Ala Ser Glu Trp Glu Arg Phe Ile Ser Lys Val Glu Glu 20 25 30 Val Leu Asn Asp Trp Lys Leu Ile Gly Asn Ser Leu Gly Lys Pro Leu 35 40 45 Glu Lys Gly Ile Phe Thr Ser Gly Thr Trp Glu Glu Lys Ser Asp Glu 50 55 60 Ile Ser Phe Ala Asp Phe Lys Phe Ser Val Thr His His Tyr Leu Val 65 70 75 80 Gln Glu Ser Thr Asp Lys Glu Gly Lys Asp Glu Leu Leu Glu Asp Val 85 90 95 Val Pro Gln Ser Met Gln Asp Leu Leu Gly Met Asn Asn Asp Phe Pro 100 105 110 Pro Arg Ala His Cys Leu Val Arg Trp Tyr Gly Leu Arg Glu Phe Val 115 120 125 Val Ile Ala Pro Ala Ala His Ser Asp Ala Val Leu Ser Glu Ser Lys 130 135 140 Cys Asn Leu Leu Leu Ser Ser Val Ser Ile Ala Leu Gly Asn Thr Gly 145 150 155 160 Cys Gln Val Pro Leu Phe Val Gln Ile His His Lys Trp Arg Arg Met 165 170 175 Tyr Val Gly Glu Cys Gln Gly Pro Gly Val Arg Thr Asp Phe Glu Met 180 185 190 Val His Leu Arg Lys Val Pro Asn Gln Tyr Thr His Leu Ser Gly Leu 195 200 205 Leu Asp Ile Phe Lys Ser Lys Ile Gly Cys Pro Leu Thr Pro Leu Pro 210 215 220 Pro Val Ser Ile Ala Ile Arg Phe Thr Tyr Val Leu Gln Asp Trp Gln 225 230 235 240 Gln Tyr Phe Trp Pro Gln Gln Pro Pro Asp Ile Asp Ala Leu Val Gly 245 250 255 Gly Glu Val Gly Gly Leu Glu Phe Gly Lys Leu Pro Phe Gly Ala Cys 260 265 270 Glu Asp Pro Ile Ser Glu Leu His Leu Ala Thr Thr Trp Pro His Leu 275 280 285 Thr Glu Gly Ile Ile Val Asp Asn Asp Val Tyr Ser Asp Leu Asp Pro 290 295 300 Ile Gln Ala Pro His Trp Ser Val Arg Val Arg Lys Ala Glu Asn Pro 305 310 315 320 Gln Cys Leu Leu Gly Asp Phe Val Thr Glu Phe Phe Lys Ile Cys Arg 325 330 335 Arg Lys Glu Ser Thr Asp Glu Ile Leu Gly Arg Ser Ala Phe Glu Glu 340 345 350 Glu Gly Lys Glu Thr Ala Asp Ile Thr His Ala Leu Ser Lys Leu Thr 355 360 365 Glu Pro Ala Ser Val Pro Ile His Lys Leu Ser Val Ser Asn Met Val 370 375 380 His Thr Ala Lys Lys Lys Ile Arg Lys His Arg Gly Val Glu Glu Ser 385 390 395 400 Pro Leu Asn Asn Asp Val Leu Asn Thr Ile Leu Leu Phe Leu Phe Pro 405 410 415 Asp Ala Val Ser Glu Lys Pro Leu Asp Gly Thr Thr Ser Thr Asp Asn 420 425 430 Asn Asn Pro Pro Ser Glu Ser Glu Asp Tyr Asn Leu Tyr Asn Gln Phe 435 440 445 Lys Ser Ala Pro Ser Asp Ser Leu Thr Tyr Lys Leu Ala Leu Cys Leu 450 455 460 Cys Met Ile Asn Phe Tyr His Gly Gly Leu Lys Gly Val Ala His Leu 465 470 475 480 Trp Gln Glu Phe Val Leu Glu Met Arg Phe Arg Trp Glu Asn Asn Phe 485 490 495 Leu Ile Pro Gly Leu Ala Ser Gly Pro Pro Asp Leu Arg Cys Cys Leu 500 505 510 Leu His Gln Lys Leu Gln Met Leu Asn Cys Cys Ile Glu Arg Lys Lys 515 520 525 Ala Arg Asp Glu Gly Lys Lys Thr Ser Ala Ser Asp Val Thr Asn Ile 530 535 540 Tyr Pro Gly Asp Ala Gly Lys Ala Gly Asp Gln Leu Val Pro Asp Asn 545 550 555 560 Leu Lys Glu Thr Asp Lys Glu Lys Gly Glu Val Gly Lys Ser Trp Asp 565 570 575 Ser Trp Ser Asp Ser Glu Glu Glu Phe Phe Glu Cys Leu Ser Asp Thr 580 585 590 Glu Glu Leu Lys Gly Asn Gly Gln Glu Ser Gly Lys Lys Gly Gly Pro 595 600 605 Lys Glu Met Ala Asn Leu Arg Pro Glu Gly Arg Leu Tyr Gln His Gly 610 615 620 Lys Leu Thr Leu Leu His Asn Gly Glu Pro Leu Tyr Ile Pro Val Thr 625 630 635 640 Gln Glu Pro Ala Pro Met Thr Glu Asp Leu Leu Glu Glu Gln Ser Glu 645 650 655 Val Leu Ala Lys Leu Gly Thr Ser Ala Glu Gly Ala His Leu Arg Ala 660 665 670 Arg Met Gln Ser Ala Cys Leu Leu Ser Asp Met Glu Ser Phe Lys Ala 675 680 685 Ala Asn Pro Gly Cys Ser Leu Glu Asp Phe Val Arg Trp Tyr Ser Pro 690 695 700 Arg Asp Tyr Ile Glu Glu Glu Val Ile Asp Glu Lys Gly Asn Val Val 705 710 715 720 Leu Lys Gly Glu Leu Ser Ala Arg Met Lys Ile Pro Ser Asn Met Trp 725 730 735 Val Glu Ala Trp Glu Thr Ala Lys Pro Ile Pro Ala Arg Arg Gln Arg 740 745 750 Arg Leu Phe Asp Asp Thr Arg Glu Ala Glu Lys Val Leu His Tyr Leu 755 760 765 Ala Ile Gln Lys Pro Ala Asp Leu Ala Arg His Leu Leu Pro Cys Val 770 775 780 Ile His Ala Ala Val Leu Lys Val Lys Glu Glu Glu Ser Leu Glu Asn 785 790 795 800 Ile Ser Ser Val Lys Lys Ile Ile Lys Gln Ile Ile Ser His Ser Ser 805 810 815 Lys Val Leu His Phe Pro Asn Pro Glu Asp Lys Lys Leu Glu Glu Ile 820 825 830 Ile His Gln Ile Thr Asn Val Glu Ala Leu Ile Ala Arg Ala Arg Ser 835 840 845 Leu Lys Ala Lys Phe Gly Thr Glu Lys Cys Glu Gln Glu Glu Glu Lys 850 855 860 Glu Asp Leu Glu Arg Phe Val Ser Cys Leu Leu Glu Gln Pro Glu Val 865 870 875 880 Leu Val Thr Gly Ala Gly Arg Gly His Ala Gly Arg Ile Ile His Lys 885 890 895 Leu Phe Val Asn Ala Gln Arg Ala Ala Ala Met Thr Pro Pro Glu Glu 900 905 910 Glu Leu Lys Arg Met Gly Ser Pro Glu Glu Arg Arg Gln Asn Ser Val 915 920 925 Ser Asp Phe Pro Pro Pro Ala Gly Arg Glu Phe Ile Leu Arg Thr Thr 930 935 940 Val Pro Arg Pro Ala Pro Tyr Ser Lys Ala Leu Pro Gln Arg Met Tyr 945 950 955 960 Ser Val Leu Thr Lys Glu Asp Phe Arg Leu Ala Gly Ala Phe Ser Ser 965 970 975 Asp Thr Ser Phe Phe 980 配列番号 : 2 配列の長さ : 2946 配列の型 : 核酸 鎖の数 : 二本鎖 トポロジー : 直鎖状 配列の種類 : cDNA to mRNA 配列の特徴 特徴を表す記号 : CDS 存在位置 : 1 .. 2943 特徴を決定した方法 : E 配 列 ATG GCT GCC GAC AGT GAG CCC GAA TCC GAG GTA TTT GAG ATC ACG GAC 48 Met Ala Ala Asp Ser Glu Pro Glu Ser Glu Val Phe Glu Ile Thr Asp 1 5 10 15 TTC ACC ACT GCC TCG GAA TGG GAA AGG TTT ATT TCC AAA GTT GAA GAA 96 Phe Thr Thr Ala Ser Glu Trp Glu Arg Phe Ile Ser Lys Val Glu Glu 20 25 30 GTC TTG AAT GAC TGG AAA CTG ATT GGA AAC TCT TTG GGA AAG CCA CTC 144 Val Leu Asn Asp Trp Lys Leu Ile Gly Asn Ser Leu Gly Lys Pro Leu 35 40 45 GAA AAG GGT ATA TTT ACT TCT GGC ACA TGG GAA GAG AAA TCA GAT GAA 192 Glu Lys Gly Ile Phe Thr Ser Gly Thr Trp Glu Glu Lys Ser Asp Glu 50 55 60 ATT TCC TTT GCT GAC TTC AAG TTC TCA GTC ACT CAT CAT TAT CTT GTA 240 Ile Ser Phe Ala Asp Phe Lys Phe Ser Val Thr His His Tyr Leu Val 65 70 75 80 CAA GAG TCC ACT GAT AAA GAA GGA AAG GAT GAG TTA TTA GAG GAT GTT 288 Gln Glu Ser Thr Asp Lys Glu Gly Lys Asp Glu Leu Leu Glu Asp Val 85 90 95 GTT CCA CAA TCT ATG CAA GAT TTG CTG GGT ATG AAT AAT GAC TTT CCT 336 Val Pro Gln Ser Met Gln Asp Leu Leu Gly Met Asn Asn Asp Phe Pro 100 105 110 CCA AGA GCA CAT TGC CTG GTA AGA TGG TAT GGG CTA CGT GAG TTC GTG 384 Pro Arg Ala His Cys Leu Val Arg Trp Tyr Gly Leu Arg Glu Phe Val 115 120 125 GTG ATT GCC CCT GCT GCA CAC AGT GAC GCT GTT CTC AGC GAA TCT AAG 432 Val Ile Ala Pro Ala Ala His Ser Asp Ala Val Leu Ser Glu Ser Lys 130 135 140 TGC AAC CTT CTT CTG AGT TCT GTT TCT ATT GCC TTG GGA AAC ACT GGC 480 Cys Asn Leu Leu Leu Ser Ser Val Ser Ile Ala Leu Gly Asn Thr Gly 145 150 155 160 TGT CAG GTG CCA CTC TTT GTG CAA ATT CAC CAC AAA TGG CGA AGA ATG 528 Cys Gln Val Pro Leu Phe Val Gln Ile His His Lys Trp Arg Arg Met 165 170 175 TAT GTA GGA GAA TGT CAA GGT CCT GGT GTA CGA ACT GAT TTC GAA ATG 576 Tyr Val Gly Glu Cys Gln Gly Pro Gly Val Arg Thr Asp Phe Glu Met 180 185 190 GTT CAT CTT AGA AAA GTG CCA AAT CAG TAC ACT CAC TTA TCA GGT CTG 624 Val His Leu Arg Lys Val Pro Asn Gln Tyr Thr His Leu Ser Gly Leu 195 200 205 CTG GAT ATC TTC AAA TCA AAG ATT GGA TGT CCT TTA ACT CCA TTG CCT 672 Leu Asp Ile Phe Lys Ser Lys Ile Gly Cys Pro Leu Thr Pro Leu Pro 210 215 220 CCA GTT AGT ATT GCT ATT CGA TTT ACC TAT GTA CTT CAA GAT TGG CAG 720 Pro Val Ser Ile Ala Ile Arg Phe Thr Tyr Val Leu Gln Asp Trp Gln 225 230 235 240 CAG TAT TTT TGG CCT CAG CAA CCT CCA GAC ATA GAT GCC CTT GTA GGA 768 Gln Tyr Phe Trp Pro Gln Gln Pro Pro Asp Ile Asp Ala Leu Val Gly 245 250 255 GGA GAA GTT GGA GGC TTG GAG TTT GGC AAG TTA CCA TTT GGT GCC TGC 816 Gly Glu Val Gly Gly Leu Glu Phe Gly Lys Leu Pro Phe Gly Ala Cys 260 265 270 GAA GAT CCT ATT AGT GAA CTC CAT TTA GCT ACT ACA TGG CCT CAT CTG 864 Glu Asp Pro Ile Ser Glu Leu His Leu Ala Thr Thr Trp Pro His Leu 275 280 285 ACC GAA GGG ATC ATT GTG GAT AAT GAT GTT TAT TCT GAT TTG GAT CCT 912 Thr Glu Gly Ile Ile Val Asp Asn Asp Val Tyr Ser Asp Leu Asp Pro 290 295 300 ATT CAA GCT CCA CAT TGG TCT GTT AGA GTT CGA AAA GCT GAG AAT CCT 960 Ile Gln Ala Pro His Trp Ser Val Arg Val Arg Lys Ala Glu Asn Pro 305 310 315 320 CAG TGT TTG CTA GGT GAT TTT GTC ACT GAA TTT TTT AAA ATT TGC CGT 1008 Gln Cys Leu Leu Gly Asp Phe Val Thr Glu Phe Phe Lys Ile Cys Arg 325 330 335 CGA AAG GAG TCA ACT GAT GAG ATT CTT GGA CGA TCT GCA TTT GAG GAA 1056 Arg Lys Glu Ser Thr Asp Glu Ile Leu Gly Arg Ser Ala Phe Glu Glu 340 345 350 GAA GGC AAA GAA ACT GCT GAT ATA ACT CAT GCT TTG TCA AAA TTG ACA 1104 Glu Gly Lys Glu Thr Ala Asp Ile Thr His Ala Leu Ser Lys Leu Thr 355 360 365 GAG CCG GCA TCA GTT CCA ATT CAT AAA TTA TCA GTT TCA AAT ATG GTA 1152 Glu Pro Ala Ser Val Pro Ile His Lys Leu Ser Val Ser Asn Met Val 370 375 380 CAC ACT GCA AAG AAG AAA ATC CGA AAA CAC AGA GGT GTA GAG GAG TCA 1200 His Thr Ala Lys Lys Lys Ile Arg Lys His Arg Gly Val Glu Glu Ser 385 390 395 400 CCG CTA AAT AAT GAT GTT CTT AAT ACT ATT CTC CTG TTC TTA TTC CCT 1248 Pro Leu Asn Asn Asp Val Leu Asn Thr Ile Leu Leu Phe Leu Phe Pro 405 410 415 GAT GCT GTT TCT GAG AAA CCA TTA GAT GGA ACT ACT TCA ACA GAT AAT 1296 Asp Ala Val Ser Glu Lys Pro Leu Asp Gly Thr Thr Ser Thr Asp Asn 420 425 430 AAT AAT CCT CCA TCA GAG AGT GAA GAC TAT AAT CTC TAC AAT CAG TTC 1344 Asn Asn Pro Pro Ser Glu Ser Glu Asp Tyr Asn Leu Tyr Asn Gln Phe 435 440 445 AAG TCT GCA CCA TCT GAC AGT TTA ACA TAC AAA CTG GCT TTG TGT CTC 1392 Lys Ser Ala Pro Ser Asp Ser Leu Thr Tyr Lys Leu Ala Leu Cys Leu 450 455 460 TGT ATG ATC AAT TTT TAC CAT GGA GGG TTG AAA GGA GTG GCA CAC CTC 1440 Cys Met Ile Asn Phe Tyr His Gly Gly Leu Lys Gly Val Ala His Leu 465 470 475 480 TGG CAG GAA TTT GTT CTT GAA ATG CGT TTC CGA TGG GAA AAC AAC TTT 1488 Trp Gln Glu Phe Val Leu Glu Met Arg Phe Arg Trp Glu Asn Asn Phe 485 490 495 CTG ATT CCA GGA TTA GCA AGT GGA CCC CCA GAT CTG AGG TGT TGT TTA 1536 Leu Ile Pro Gly Leu Ala Ser Gly Pro Pro Asp Leu Arg Cys Cys Leu 500 505 510 CTG CAT CAG AAA CTA CAG ATG TTA AAT TGT TGT ATT GAA AGA AAG AAG 1584 Leu His Gln Lys Leu Gln Met Leu Asn Cys Cys Ile Glu Arg Lys Lys 515 520 525 GCA CGT GAT GAG GGG AAA AAG ACA AGT GCT TCA GAT GTC ACT AAT ATA 1632 Ala Arg Asp Glu Gly Lys Lys Thr Ser Ala Ser Asp Val Thr Asn Ile 530 535 540 TAT CCA GGG GAT GCT GGA AAA GCA GGA GAC CAG TTG GTG CCA GAT AAT 1680 Tyr Pro Gly Asp Ala Gly Lys Ala Gly Asp Gln Leu Val Pro Asp Asn 545 550 555 560 CTA AAA GAA ACA GAT AAG GAA AAG GGA GAG GTA GGA AAA TCT TGG GAT 1728 Leu Lys Glu Thr Asp Lys Glu Lys Gly Glu Val Gly Lys Ser Trp Asp 565 570 575 TCC TGG AGT GAC AGC GAA GAA GAA TTT TTT GAA TGC CTA AGT GAT ACT 1776 Ser Trp Ser Asp Ser Glu Glu Glu Phe Phe Glu Cys Leu Ser Asp Thr 580 585 590 GAA GAA CTT AAA GGA AAT GGA CAA GAG AGT GGC AAG AAA GGA GGA CCT 1824 Glu Glu Leu Lys Gly Asn Gly Gln Glu Ser Gly Lys Lys Gly Gly Pro 595 600 605 AAG GAG ATG GCA AAT TTA AGG CCG GAA GGA CGG CTC TAT CAG CAT GGG 1872 Lys Glu Met Ala Asn Leu Arg Pro Glu Gly Arg Leu Tyr Gln His Gly 610 615 620 AAA CTT ACA CTG CTG CAT AAT GGA GAA CCT CTC TAC ATT CCA GTA ACC 1920 Lys Leu Thr Leu Leu His Asn Gly Glu Pro Leu Tyr Ile Pro Val Thr 625 630 635 640 CAG GAA CCA GCA CCT ATG ACA GAA GAT CTG CTA GAA GAG CAG TCT GAA 1968 Gln Glu Pro Ala Pro Met Thr Glu Asp Leu Leu Glu Glu Gln Ser Glu 645 650 655 GTT TTA GCT AAA TTA GGT ACA TCG GCA GAG GGG GCT CAC CTT CGA GCA 2016 Val Leu Ala Lys Leu Gly Thr Ser Ala Glu Gly Ala His Leu Arg Ala 660 665 670 CGC ATG CAG AGT GCC TGT CTG CTC TCA GAT ATG GAG TCT TTT AAG GCA 2064 Arg Met Gln Ser Ala Cys Leu Leu Ser Asp Met Glu Ser Phe Lys Ala 675 680 685 GCT AAT CCA GGT TGC TCC CTG GAA GAT TTT GTG AGG TGG TAT TCA CCC 2112 Ala Asn Pro Gly Cys Ser Leu Glu Asp Phe Val Arg Trp Tyr Ser Pro 690 695 700 CGG GAT TAT ATT GAA GAG GAG GTG ATT GAT GAA AAG GGC AAT GTG GTG 2160 Arg Asp Tyr Ile Glu Glu Glu Val Ile Asp Glu Lys Gly Asn Val Val 705 710 715 720 CTG AAA GGA GAA CTG AGT GCC CGG ATG AAG ATT CCA AGC AAT ATG TGG 2208 Leu Lys Gly Glu Leu Ser Ala Arg Met Lys Ile Pro Ser Asn Met Trp 725 730 735 GTA GAA GCC TGG GAA ACA GCT AAG CCA ATT CCT GCT AGA AGG CAA AGG 2256 Val Glu Ala Trp Glu Thr Ala Lys Pro Ile Pro Ala Arg Arg Gln Arg 740 745 750 AGA CTC TTT GAT GAT ACA CGG GAA GCA GAA AAG GTG CTG CAC TAT CTG 2304 Arg Leu Phe Asp Asp Thr Arg Glu Ala Glu Lys Val Leu His Tyr Leu 755 760 765 GCA ATC CAG AAA CCT GCA GAC CTT GCT CGG CAC CTG TTA CCT TGT GTG 2352 Ala Ile Gln Lys Pro Ala Asp Leu Ala Arg His Leu Leu Pro Cys Val 770 775 780 ATT CAT GCA GCT GTA CTC AAG GTA AAG GAA GAA GAA AGT CTC GAA AAC 2400 Ile His Ala Ala Val Leu Lys Val Lys Glu Glu Glu Ser Leu Glu Asn 785 790 795 800 ATT TCT TCA GTT AAG AAG ATC ATA AAG CAG ATA ATA TCC CAT TCC AGT 2448 Ile Ser Ser Val Lys Lys Ile Ile Lys Gln Ile Ile Ser His Ser Ser 805 810 815 AAA GTT TTG CAC TTC CCC AAT CCA GAA GAC AAG AAA TTG GAA GAA ATC 2496 Lys Val Leu His Phe Pro Asn Pro Glu Asp Lys Lys Leu Glu Glu Ile 820 825 830 ATT CAC CAG ATT ACT AAT GTG GAA GCT CTC ATT GCC AGA GCT CGG TCA 2544 Ile His Gln Ile Thr Asn Val Glu Ala Leu Ile Ala Arg Ala Arg Ser 835 840 845 CTA AAA GCC AAG TTT GGA ACT GAG AAA TGT GAA CAG GAG GAG GAA AAG 2592 Leu Lys Ala Lys Phe Gly Thr Glu Lys Cys Glu Gln Glu Glu Glu Lys 850 855 860 GAA GAT CTT GAA AGG TTT GTG AGT TGC CTG CTG GAG CAG CCT GAA GTG 2640 Glu Asp Leu Glu Arg Phe Val Ser Cys Leu Leu Glu Gln Pro Glu Val 865 870 875 880 TTA GTC ACC GGT GCA GGA AGA GGA CAT GCT GGC AGG ATC ATT CAC AAG 2688 Leu Val Thr Gly Ala Gly Arg Gly His Ala Gly Arg Ile Ile His Lys 885 890 895 CTG TTT GTG AAT GCC CAG AGG GCT GCA GCT ATG ACT CCA CCA GAG GAG 2736 Leu Phe Val Asn Ala Gln Arg Ala Ala Ala Met Thr Pro Pro Glu Glu 900 905 910 GAA TTG AAG AGA ATG GGC TCC CCA GAG GAA AGA AGG CAG AAC TCC GTG 2784 Glu Leu Lys Arg Met Gly Ser Pro Glu Glu Arg Arg Gln Asn Ser Val 915 920 925 TCA GAC TTC CCA CCC CCT GCT GGC CGG GAA TTC ATT TTG CGC ACC ACT 2832 Ser Asp Phe Pro Pro Pro Ala Gly Arg Glu Phe Ile Leu Arg Thr Thr 930 935 940 GTG CCG CGC CCT GCT CCC TAC TCC AAA GCT CTG CCT CAG CGG ATG TAC 2880 Val Pro Arg Pro Ala Pro Tyr Ser Lys Ala Leu Pro Gln Arg Met Tyr 945 950 955 960 AGT GTT CTC ACC AAA GAG GAC TTT AGA CTT GCA GGT GCC TTT TCA TCA 2928 Ser Val Leu Thr Lys Glu Asp Phe Arg Leu Ala Gly Ala Phe Ser Ser 965 970 975 GAT ACT TCC TTC TTC TGA 2946 Asp Thr Ser Phe Phe 980 配列番号：3 配列の長さ：19 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Pro Val Pro Ala Arg Arg Gln Arg Arg Leu Phe Asp Asp Thr Arg Glu Ala Glu 1 5 10 15 Lys 配列番号：4 配列の長さ：11 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Leu Thr Glu Pro Ala Pro Val Pro Ile His Lys 1 5 10 配列番号：5 配列の長さ：16 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Asp Met Ala Pro Leu Lys Pro Glu Gly Arg Leu His Gln His Gly Lys 1 5 10 15 配列番号：6 配列の長さ：10 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Ala Ala Asp Ser Glu Pro Glu Ser Glu Val 1 5 10 SEQ ID NO: 1 Sequence length: 981 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Ala Ala Asp Ser Glu Pro Glu Ser Glu Val Phe Glu Ile Thr Asp 1 5 10 15 Phe Thr Thr Ala Ser Glu Trp Glu Arg Phe Ile Ser Lys Val Glu Glu 20 25 30 Val Leu Asn Asp Trp Lys Leu Ile Gly Asn Ser Leu Gly Lys Pro Leu 35 40 45 Glu Lys Gly Ile Phe Thr Ser Gly Thr Trp Glu Glu Lys Ser Asp Glu 50 55 60 Ile Ser Phe Ala Asp Phe Lys Phe Ser Val Thr His His Tyr Leu Val 65 70 75 80 Gln Glu Ser Thr Asp Lys Glu Gly Lys Asp Glu Leu Leu Glu Asp Val 85 90 95 Val Pro Gln Ser Met Gln Asp Leu Leu Gly Met Asn Asn Asp Phe Pro 100 105 110 Pro Arg Ala His Cys Leu Val Arg Trp Tyr Gly Leu Arg Glu Phe Val 115 120 125 Val Ile Ala Pro Ala Ala His Ser Asp Ala Val Leu Ser Glu Ser Lys 130 135 140 Cys Asn Leu Leu Leu Ser Ser Val Ser Ile Ala Leu Gly Asn Thr Gly 145 150 155 160 Cys Gln Val Pro Leu Phe Val Gln Ile His His Lys Trp Arg Arg Met 165 170 175 Tyr Val Gly Glu Cys Gln Gly Pro Gly Val Arg Thr Asp Phe Glu Met 180 185 190 Val His Leu Arg Lys Val Pro Asn Gln Tyr Thr His Leu Ser Gly Leu 195 200 205 Leu Asp Ile Phe Lys Ser Lys Ile Gly Cys Pro Leu Thr Pro Leu Pro 210 215 220 Pro Val Ser Ile Ala Ile Arg Phe Thr Tyr Val Leu Gln Asp Trp Gln 225 230 235 240 Gln Tyr Phe Trp Pro Gln Gln Pro Pro Asp Ile Asp Ala Leu Val Gly 245 250 255 Gly Glu Val Gly Gly Leu Glu Phe Gly Lys Leu Pro Phe Gly Ala Cys 260 265 270 Glu Asp Pro Ile Ser Glu Leu His Leu Ala Thr Thr Trp Pro His Leu 275 280 285 Thr Glu Gly Ile Ile Val Asp Asn Asp Val Tyr Ser Asp Leu Asp Pro 290 295 300 Ile Gln Ala Pro His Trp Ser Val Arg Val Arg Lys Ala Glu Asn Pro 305 310 315 320 Gln Cys Leu Leu Gly Asp Phe Val Thr Glu Phe Phe Lys Ile Cys Arg 325 330 335 Arg Lys Glu Ser Thr Asp Glu Ile Leu Gly Arg Ser Ala Phe Glu Glu 340 345 350 Glu Gly Lys Glu Thr Ala Asp Ile Thr His Ala Leu Ser Lys Leu Thr 355 360 365 Glu Pro Ala Ser Val Pro Ile His Lys Leu Ser Val Ser Asn Met Val 370 375 380 His Thr Ala Lys Lys Lys Ile Arg Lys HisArg Gly Val Glu Glu Ser 385 390 395 400 Pro Leu Asn Asn Asp Val Leu Asn Thr Ile Leu Leu Phe Leu Phe Pro 405 410 415 Asp Ala Val Ser Glu Lys Pro Leu Asp Gly Thr Thr Ser Thr Asp Asn 420 425 430 Asn Asn Pro Pro Ser Glu Ser Glu Asp Tyr Asn Leu Tyr Asn Gln Phe 435 440 445 Lys Ser Ala Pro Ser Asp Ser Leu Thr Tyr Lys Leu Ala Leu Cys Leu 450 455 460 Cys Met Ile Asn Phe Tyr His Gly Gly Leu Lys Gly Val Ala His Leu 465 470 475 480 Trp Gln Glu Phe Val Leu Glu Met Arg Phe Arg Trp Glu Asn Asn Phe 485 490 495 Leu Ile Pro Gly Leu Ala Ser Gly Pro Pro Asp Leu Arg Cys Cys Leu 500 505 510 Leu His Gln Lys Leu Gln Met Leu Asn Cys Cys Ile Glu Arg Lys Lys 515 520 525 Ala Arg Asp Glu Gly Lys Lys Thr Ser Ala Ser Asp Val Thr Asn Ile 530 535 540 Tyr Pro Gly Asp Ala Gly Lys Ala Gly Asp Gln Leu Val Pro Asp Asn 545 550 555 560 Leu Lys Glu Thr Asp Lys Glu Lys Gly Glu Val Gly Lys Ser Trp Asp 565 570 575 Ser Trp Ser Asp Ser Glu Glu Glu Phe Phe Glu Cys Leu Ser Asp Thr 580 585 590 Glu Glu Glu Leu Lys Gly Asn Gly Gln Glu Ser Gly Lys Lys Gly Gly Pro 595 600 605 Lys Glu Met Ala Asn Leu Arg Pro Glu Gly Arg Leu Tyr Gln His Gly 610 615 620 Lys Leu Thr Leu Leu His Asn Gly Glu Pro Leu Tyr Ile Pro Val Thr 625 630 630 635 640 Gln Glu Pro Ala Pro Met Thr Glu Asp Leu Leu Glu Glu Gln Ser Glu 645 650 655 Val Leu Ala Lys Leu Gly Thr Ser Ala Glu Gly Ala His Leu Arg Ala 660 665 670 Arg Met Gln Ser Ala Cys Leu Leu Ser Asp Met Glu Ser Phe Lys Ala 675 680 685 Ala Asn Pro Gly Cys Ser Leu Glu Asp Phe Val Arg Trp Tyr Ser Pro 690 695 700 Arg Asp Tyr Ile Glu Glu Glu Glu Val Ile Asp Glu Lys Gly Asn Val Val 705 710 710 715 720 Leu Lys Gly Glu Leu Ser Ala Arg Met Lys Ile Pro Ser Asn Met Trp 725 730 735 735 Val Glu Ala Trp Glu Thr Ala Lys Pro Ile Pro Ala Arg Arg Gln Arg 740 745 750 Arg Leu Phe Asp Asp Thr Arg Glu Ala Glu Lys Val Leu His Tyr Leu 755 760 765 Ala Ile Gln Lys Pro Ala Asp Leu Ala Arg His Leu Leu Pro Cys Val 770 775 775 780 Ile His Ala Ala Val Leu Lys Val Lys Glu Glu Glu Glu Ser Leu Glu Asn 785 790 795 800 Ile Ser Ser Val Lys Lys Ile Ile Lys Gln Ile Ile Ser His Ser Ser 805 810 815 Lys Val Leu His Phe Pro Asn Pro Glu Asp Lys Lys Leu Glu Glu Ile 820 825 830 Ile His Gln Ile Thr Asn Val Glu Ala Leu Ile Ala Arg Ala Arg Ser 835 840 845 Leu Lys Ala Lys Phe Gly Thr Glu Lys Cys Glu Gln Glu Glu Glu Lys 850 855 860 Glu Asp Leu Glu Arg Phe Val Ser Cys Leu Leu Glu Gln Pro Glu Val 865 870 875 880 Leu Val Thr Gly Ala Gly Arg Gly His Ala Gly Arg Ile Ile His Lys 885 890 895 Leu Phe Val Asn Ala Gln Arg Ala Ala Ala Met Thr Pro Pro Glu Glu 900 905 910 Glu Leu Lys Arg Met Gly Ser Pro Glu Glu Arg Arg Gln Asn Ser Val 915 920 925 925 Ser Asp Phe Pro Pro Pro Ala Gly Arg Glu Phe Ile Leu Arg Thr Thr 930 935 940 Val Pro Arg Pro Ala Pro Tyr Ser Lys Ala Leu Pro Gln Arg Met Tyr 945 950 955 960 Ser Val Leu Thr Lys Glu Asp Phe Arg Leu Ala Gly Ala Phe Ser Ser 965 970 975 Asp Thr Ser Phe Phe 980 SEQ ID NO: 2 Sequence length: 2946 Sequence type: Nucleic acid number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence characteristics Features Symbol: CDS Location: 1 .. 2943 Method for determining characteristics: E sequence ATG GCT GCC GAC AGT GAG CCC GAA TCC GAG GTA TTT GAG ATC ACG GAC 48 Met Ala Ala Asp Ser Glu Pro Glu Ser Glu Val Phe Glu Ile Thr Asp 1 5 10 15 TTC ACC ACT GCC TCG GAA TGG GAA AGG TTT ATT TCC AAA GTT GAA GAA 96 Phe Thr Thr Ala Ser Glu Trp Glu Arg Phe Ile Ser Lys Val Glu Glu 20 25 30 GTC TTG AAT GAC TGG AAA CTG ATT GGA AAC TCT TTG GGA AAG CCA CTC 144 Val Leu Asn Asp Trp Lys Leu Ile Gly Asn Ser Leu Gly Lys Pro Leu 35 40 45 GAA AAG GGT ATA TTT ACT TCT GGC ACA TGG GAA GAG AAA TCA GAT GAA 192 Glu Lys Gly Ile Phe Thr Ser Gly Thr Trp Glu Glu Lys Ser Asp Glu 50 55 60 ATT TCC TTT GCT GAC TTC AAG TTC TCA GTC ACT CAT CAT TAT CTT GTA 240 Ile Ser Phe Ala Asp Phe Lys Phe Ser Val Thr His His Tyr Leu Val 65 70 75 80 CAA GAG TCC ACT GAT AAA GAA GGA AAG GAT GAG TTA TTA GAG GAT GTT 288 Gln Glu Ser Thr Asp Lys Glu Gly Lys Asp Glu Leu Leu Glu Asp Val 85 90 95 GTT CCA CAA TCT ATG CAA GAT TTG CTG GGT ATG AAT AAT GAC TTT CCT 336 V al Pro Gln Ser Met Gln Asp Leu Leu Gly Met Asn Asn Asp Phe Pro 100 105 110 CCA AGA GCA CAT TGC CTG GTA AGA TGG TAT GGG CTA CGT GAG TTC GTG 384 Pro Arg Ala His Cys Leu Val Arg Trp Tyr Gly Leu Arg Glu Phe Val 115 120 125 GTG ATT GCC CCT GCT GCA CAC AGT GAC GCT GTT CTC AGC GAA TCT AAG 432 Val Ile Ala Pro Ala Ala His Ser Asp Ala Val Leu Ser Glu Ser Lys 130 135 140 TGC AAC CTT CTT CTG AGT TCT GTT TCT ATT GCC TTG GGA AAC ACT GGC 480 Cys Asn Leu Leu Leu Ser Ser Val Ser Ile Ala Leu Gly Asn Thr Gly 145 150 155 160 TGT CAG GTG CCA CTC TTT GTG CAA ATT CAC CAC AAA TGG CGA AGA ATG 528 Cys Gln Val Pro Leu Phe Val Gln Ile His His Lys Trp Arg Arg Met 165 170 175 TAT GTA GGA GAA TGT CAA GGT CCT GGT GTA CGA ACT GAT TTC GAA ATG 576 Tyr Val Gly Glu Cys Gln Gly Pro Gly Val Arg Thr Asp Phe Glu Met 180 185 190 GTT CAT CTT AGA AAA GTG CCA AAT CAG TAC ACT CAC TTA TCA GGT CTG 624 Val His Leu Arg Lys Val Pro Asn Gln Tyr Thr His Leu Ser Gly Leu 195 200 205 CTG GAT ATC TTC AAA TCA AAG ATT GGA TGT CCT TTA ACT CCA T TG CCT 672 Leu Asp Ile Phe Lys Ser Lys Ile Gly Cys Pro Leu Thr Pro Leu Pro 210 215 220 CCA GTT AGT ATT GCT ATT CGA TTT ACC TAT GTA CTT CAA GAT TGG CAG 720 Pro Val Ser Ile Ala Ile Arg Phe Thr Tyr Val Leu Gln Asp Trp Gln 225 230 235 240 CAG TAT TTT TGG CCT CAG CAA CCT CCA GAC ATA GAT GCC CTT GTA GGA 768 Gln Tyr Phe Trp Pro Gln Gln Pro Pro Asp Ile Asp Ala Leu Val Gly 245 250 255 GGA GAA GTT GGA GGC TTG GAG TTT GGC AAG TTA CCA TTT GGT GCC TGC 816 Gly Glu Val Gly Gly Leu Glu Phe Gly Lys Leu Pro Phe Gly Ala Cys 260 265 270 GAA GAT CCT ATT AGT GAA CTC CAT TTA GCT ACT ACA TGG CCT CAT CTG 864 Glu Asp Pro Ile Ser Glu Leu His Leu Ala Thr Thr Trp Pro His Leu 275 280 285 ACC GAA GGG ATC ATT GTG GAT AAT GAT GTT TAT TCT GAT TTG GAT CCT 912 Thr Glu Gly Ile Ile Val Asp Asn Asp Val Tyr Ser Asp Leu Asp Pro 290 295 300 ATT CAA GCT CCA CAT TGG TCT GTT AGA GTT CGA AAA GCT GAG AAT CCT 960 Ile Gln Ala Pro His Trp Ser Val Arg Val Arg Lys Ala Glu Asn Pro 305 310 315 320 CAG TGT TTG CTA GGT GAT TTT GTC ACT GAA T TT TTT AAA ATT TGC CGT 1008 Gln Cys Leu Leu Gly Asp Phe Val Thr Glu Phe Phe Lys Ile Cys Arg 325 330 335 CGA AAG GAG TCA ACT GAT GAG ATT CTT GGA CGA TCT GCA TTT GAG GAA 1056 Arg Lys Glu Ser Thr Asp Glu Ile Leu Gly Arg Ser Ala Phe Glu Glu 340 345 350 GAA GGC AAA GAA ACT GCT GAT ATA ACT CAT GCT TTG TCA AAA TTG ACA 1104 Glu Gly Lys Glu Thr Ala Asp Ile Thr His Ala Leu Ser Lys Leu Thr 355 360 365 GAG CCG GCA TCA GTT CCA ATT CAT AAA TTA TCA GTT TCA AAT ATG GTA 1152 Glu Pro Ala Ser Val Pro Ile His Lys Leu Ser Val Ser Asn Met Val 370 375 380 CAC ACT GCA AAG AAG AAA ATC CGA AAA CAC AGA GGT GTA GAG GAG TCA 1200 His Thr Ala Lys Lys Lys Ile Arg Lys His Arg Gly Val Glu Glu Ser 385 390 395 400 400 CCG CTA AAT AAT GAT GTT CTT AAT ACT ATT CTC CTG TTC TTA TTC CCT 1248 Pro Leu Asn Asn Asp Val Leu Asn Thr Ile Leu Leu Phe Leu Phe Pro 405 410 415 GAT GCT GTT TCT GAG AAA CCA TTA GAT GGA ACT ACT TCA ACA GAT AAT 1296 Asp Ala Val Ser Glu Lys Pro Leu Asp Gly Thr Thr Ser Thr Asp Asn 420 425 430 AAT AAT CCT CCA TCA G AG AGT GAA GAC TAT AAT CTC TAC AAT CAG TTC 1344 Asn Asn Pro Pro Ser Glu Ser Glu Asp Tyr Asn Leu Tyr Asn Gln Phe 435 440 445 AAG TCT GCA CCA TCT GAC AGT TTA ACA TAC AAA CTG GCT TTG TGT CTC 1392 Lys Ser Ala Pro Ser Asp Ser Leu Thr Tyr Lys Leu Ala Leu Cys Leu 450 455 460 TGT ATG ATC AAT TTT TAC CAT GGA GGG TTG AAA GGA GTG GCA CAC CTC 1440 Cys Met Ile Asn Phe Tyr His Gly Gly Leu Lys Gly Val Ala His Leu 465 470 475 480 TGG CAG GAA TTT GTT CTT GAA ATG CGT TTC CGA TGG GAA AAC AAC TTT 1488 Trp Gln Glu Phe Val Leu Glu Met Arg Phe Arg Trp Glu Asn Asn Phe 485 490 495 CTG ATT CCA GGA TTA GCA AGT GGACC C GAT CTG AGG TGT TGT TTA 1536 Leu Ile Pro Gly Leu Ala Ser Gly Pro Pro Asp Leu Arg Cys Cys Leu 500 505 510 CTG CAT CAG AAA CTA CAG ATG TTA AAT TGT TGT ATT GAA AGA AAG AAG 1584 Leu His Gln Lys Leu Gln Met Leu Asn Cys Cys Ile Glu Arg Lys Lys 515 520 525 GCA CGT GAT GAG GGG AAA AAG ACA AGT GCT TCA GAT GTC ACT AAT ATA 1632 Ala Arg Asp Glu Gly Lys Lys Thr Ser Ala Ser Asp Val Thr Asn Ile 530 535 540 TA T CCA GGG GAT GCT GGA AAA GCA GGA GAC CAG TTG GTG CCA GAT AAT 1680 Tyr Pro Gly Asp Ala Gly Lys Ala Gly Asp Gln Leu Val Pro Asp Asn 545 550 555 555 560 CTA AAA GAA ACA GAT AAG GAA AAG GGA GAG GTA GGA AAA TCT TGG GAT 1728 Leu Lys Glu Thr Asp Lys Glu Lys Gly Glu Val Gly Lys Ser Trp Asp 565 570 575 TCC TGG AGT GAC AGC GAA GAA GAA TTT TTT GAA TGC CTA AGT GAT ACT 1776 Ser Trp Ser Asp Ser Glu Glu Glu Plu Phe Phe Glu Cys Leu Ser Asp Thr 580 585 590 GAA GAA CTT AAA GGA AAT GGA CAA GAG AGT GGC AAG AAA GGA GGA CCT 1824 Glu Glu Leu Lys Gly Asn Gly Gln Glu Ser Gly Lys Lys Gly Gly Pro 595 600 605 AAG GAG ATG GCA AAT TTA AGG CCG GAA GGA CGG CTC TAT CAG CAT GGG 1872 Lys Glu Met Ala Asn Leu Arg Pro Glu Gly Arg Leu Tyr Gln His Gly 610 615 620 AAA CTT ACA CTG CTG CAT AAT GGA GAA CCT CTC TAC ATT CCA GTA ACC 1920 Lys Leu Thr Leu Leu His Asn Gly Glu Pro Leu Tyr Ile Pro Val Thr 625 630 635 640 CAG GAA CCA GCA CCT ATG ACA GAA GAT CTG CTA GAA GAG CAG TCT GAA 1968 Gln Glu Pro Ala Pro Met Thr Glu Asp Leu Leu Glu Glu Glu Gln Ser Glu 645 650 655 GTT TTA GCT AAA TTA GGT ACA TCG GCA GAG GGG GCT CAC CTT CGA GCA 2016 Val Leu Ala Lys Leu Gly Thr Ser Ala Glu Gly Ala His Leu Arg Ala 660 665 670 670 CGC ATG CAG AGT GCC TGT CTG CTC TCA GAT ATG GAG TCT TTT AAG GCA 2064 Arg Met Gln Ser Ala Cys Leu Leu Ser Asp Met Glu Ser Phe Lys Ala 675 680 685 GCT AAT CCA GGT TGC TCC CTG GAA GAT TTT GTG AGG TGG TAT TCA CCC 2112 Ala Asn Pro Gly Cys Ser Leu Glu Asp Phe Val Arg Trp Tyr Ser Pro 690 695 700 CGG GAT TAT ATT GAA GAG GAG GTG ATT GAT GAA AAG GGC AAT GTG GTG 2160 Arg Asp Tyr Ile Glu Glu Glu Val Ile Asp Glu Lys Gly Asn Val Val 705 710 710 715 715 720 CTG AAA GGA GAA CTG AGT GCC CGG ATG AAG ATT CCA AGC AAT ATG TGG 2208 Leu Lys Gly Glu Leu Ser Ala Arg Met Lys Ile Pro Ser Asn Met Trp 725 730 735 735 GTA GAA GCC TGG GAA ACA GCT AAG CCA ATT CCT GCT AGA AGG CAA AGG 2256 Val Glu Ala Trp Glu Thr Ala Lys Pro Ile Pro Ala Arg Arg Gln Arg 740 745 750 AGA CTC TTT GAT GAT ACA CGG GAA GCA GAA AAG GTG CTG CAC TAT CTG 2304 Arg Leu Phe Asp Asp Thr Arg Glu Ala Glu Lys Val Leu His Tyr Leu 755 760 765 GCA ATC CAG AAA CCT GCA GAC CTT GCT CGG CAC CTG TTA CCT TGT GTG 2352 Ala Ile Gln Lys Pro Ala Asp Leu Ala Arg His Leu Leu Pro Cys Val 770 775 775 780 ATT CAT GCA GCT GTA CTC AAG GTA AAG GAA GAA GAA AGT CTC GAA AAC 2400 Ile His Ala Ala Val Leu Lys Val Lys Glu Glu Glu Ser Leu Glu Asn 785 790 795 800 ATT TCT TCA GTT AAG AAG ATC ATA AAG CAG ATA ATA TCC CAT TCC AGT 2448 Ile Ser Ser Val Lys Lys Ile Ile Lys Gln Ile Ile Ser His Ser Ser 805 810 815 AAA GTT TTG CAC TTC CCC AAT CCA GAA GAC AAG AAA TTG GAA GAA ATC 2496 Lys Val Leu His Phe Pro Asn Pro Glu Asp Lys Lys Leu Glu Glu Ile 820 825 830 ATT CAC CAG ATT ACT AAT GTG GAA GCT CTC ATT GCC AGA GCT CGG TCA 2544 Ile His Gln Ile Thr Asn Val Glu Ala Leu Ile Ala Arg Ala Arg Ser 835 840 845 845 CTA AAA GCC AAG TTT GGA ACT GAG AAA TGT GAA CAG GAG GAG GAA AAG 2592 Leu Lys Ala Lys Phe Gly Thr Glu Lys Cys Glu Gln Glu Glu Glu Lys 850 855 860 GAA GAT CTT GAA AGG TTT GTG AGT TGC CTG CTG GAG CAG CCT GAA GTG 2640 Glu Asp Leu Glu Arg Phe Val Ser Cys Leu Leu Glu Gln Pro Glu Val 865 870 875 880 TTA GTC ACC GGT GCA GGA AGA GGA CAT GCT GGC AGG ATC ATT CAC AAG 2688 Leu Val Thr Gly Ala Gly Arg Gly His Ala Gly Arg Ile Ile His Lys 885 890 895 CTG TTT GTG AAT GCC CAG AGG GCT GCA GCT ATG ACT CCA CCA GAG GAG 2736 Leu Phe Val Asn Ala Gln Arg Ala Ala Ala Met Thr Pro Pro Glu Glu 900 905 910 GAA TTG AAG AGA ATG GGC TCC CCA GAG GAA AGA AGG CAG AAC TCC GTG 2784 Glu Leu Lys Arg Met Gly Ser Pro Glu Glu Arg Arg Gln Asn Ser Val 915 920 925 TCA GAC TTC CCA CCC CCT GCT GGC CGG GAA TTC ATT TTG CGC ACC ACT 2832 Ser Asp Phe Pro Pro Pro Ala Gly Arg Glu Phe Ile Leu Arg Thr Thr 930 935 940 GTG CCG CGC CCT GCT CCC TAC TCC AAA GCT CTG CCT CAG CGG ATG TAC 2880 Val Pro Arg Pro Ala Pro Tyr Ser Lys Ala Leu Pro Gln Arg Met Tyr 945 950 955 960 AGT GTT CTC ACC AAA GAG GAC TTT AGA CTT GCA GGT GCC TTT TCA TCA 2928 Ser Val Leu Thr Lys Glu Asp Phe Arg Leu Ala Gly Ala Phe Ser Ser 965 970 975 GAT ACT TCC TTC TTC TGA 2946 Asp Thr Ser Phe Phe 980 Sequence No .: 3 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Pro Val Pro Ala Arg Arg Gln Arg Arg Leu Phe Asp Asp Thr Arg Glu Ala Glu 1 5 10 15 Lys SEQ ID NO: : 4 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Thr Glu Pro Ala Pro Val Pro Ile His Lys 1 5 10 SEQ ID NO: 5 Sequence length: 16 Sequence Type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Met Ala Pro Leu Lys Pro Glu Gly Arg Leu His Gln His Gly Lys 1 5 10 15 SEQ ID NO: 6 Sequence length: 10 Sequence type: Amino acid topology : Linear Sequence type: Peptide sequence Ala Ala Asp Ser Glu Pro Glu Ser Glu Val 1 5 10
【図１】AはモノQカラムクロマトグラフィーでのRab3GA
P活性の溶出のプロフィールを示す図である。基質とし
て[γ-32P]GTP-Rab3Aを用いて各分画の一部(0.25μl)の
Rab3GAP活性を測定した。(●)はフィルター上の残留放
射活性を、(---)は280nmの吸光度を、（−）はNaCl濃度
をそれぞれ示す。BはモノQカラムクロマトグラフィーの
タンパク質染色像である。各分画の一部(5μl)をSDS-PA
GE(8％ポリアクリルアミドゲル)にかけ、続いてタンパ
ク質を銀染色した。Cはスクロース密度勾配超遠心でのR
ab3GAP活性の溶出プロフィール及びタンパク質染色像を
示す。モノQPC1.6/5分画のサンプルを「緩衝液A」を含
む4.8ml直線スクロース勾配(5-40％)に重層し、219,000
gで13.8時間遠心した。160μlずつの分画を集めた。各
分画の一部(20μl)をSDS-PAGEにかけ、続いてタンパク
質を銀染色した。(●)はフィルター上の残留放射活性を
示し、挿入図はタンパク質染色像を示す。Dは再び行っ
たモノQカラムクロマトグラフィーでのRab3GAP活性の溶
出プロフィール及びタンパク質染色像を示す。スクロー
ス密度勾配超遠心のサンプル(450μl)を集め、1.2% CHA
PS及び1350μlの「緩衝液C」(0.6%CHAPSを含む緩衝液A)
を含む緩衝液A450μlで希釈した。サンプルを「緩衝液
C」とともにモノQ PC1.6/5カラムクロマトグラフィーに
て分画した。カラムを2mlの280mM NaClを含む「緩衝液
C」で洗浄後、3mlのNaCl直線勾配(280-500mM)を用いて
溶出を行い、続いて0.5mlのNaCl直線勾配(0.5-1M)、及
び「緩衝液C」中の1M NaClを流出速度0.1ml/分で行い、
100μlずつの分画を集めた。各分画別の一部(60μl)をS
DS-PAGEにかけ、続いて銀染色した。(●)はフィルター
上の残留放射活性を示し、挿入図はタンパク質染色像を
示す。Fig. A: Rab3GA by mono-Q column chromatography
FIG. 3 is a view showing a profile of elution of P activity. Using [γ- 32 P] GTP-Rab3A as a substrate, a portion (0.25 μl) of each fraction
Rab3GAP activity was measured. (●) indicates the residual radioactivity on the filter, (---) indicates the absorbance at 280 nm, and (-) indicates the NaCl concentration. B is a protein staining image of Mono Q column chromatography. A portion (5 μl) of each fraction was SDS-PA
The proteins were run on GE (8% polyacrylamide gel) followed by silver staining. C is R in sucrose density gradient ultracentrifugation
1 shows the elution profile and protein staining image of ab3GAP activity. A sample of the mono QPC 1.6 / 5 fraction was overlaid on a 4.8 ml linear sucrose gradient (5-40%) containing "Buffer A" and 219,000
Centrifuged at g for 13.8 hours. 160 μl fractions were collected. A portion (20 μl) of each fraction was subjected to SDS-PAGE, followed by silver staining of the protein. (●) indicates the residual radioactivity on the filter, and the inset shows the protein staining image. D shows the elution profile of Rab3GAP activity and the protein staining image in the mono Q column chromatography performed again. Collect a sucrose gradient ultracentrifuge sample (450 μl) and add 1.2% CHA
PS and 1350 μl of `` buffer C '' (buffer A containing 0.6% CHAPS)
Was diluted with 450 μl of buffer A containing. Samples in buffer
C and Mono Q PC1.6 / 5 column chromatography. Columns containing 2 ml of 280 mM NaCl
`` C '', elution was carried out with a 3 ml linear gradient of NaCl (280-500 mM), followed by a 0.5 ml linear gradient of NaCl (0.5-1 M) and the efflux rate of 1 M NaCl in `` buffer C '' Perform at 0.1 ml / min,
Fractions of 100 μl were collected. A portion (60 μl) of each fraction
DS-PAGE followed by silver staining. (●) indicates the residual radioactivity on the filter, and the inset shows the protein staining image.
【図２】AはOverlay法にてRab3GAP活性を検出するため
に用いたモノQサンプルの電気泳動像である。Bは薄層ク
ロマトグラフィーにてRab3GAP活性を検出した結果を示
す図である。[α-32P]GTP-Rab3AをRAb3GAPのモノQサン
プル(50ng)の存在下または非存在下で、30℃で5分間イ
ンキュベートし、続いてフィルターに通した。Rab3Aに
結合したグアニンヌクレオチドをフィルターから溶出
し、溶出されたヌクレオチドを薄層クロマトグラフィー
により分離した。FIG. 2A is an electrophoretic image of a mono-Q sample used for detecting Rab3GAP activity by the overlay method. B is a diagram showing the results of detecting Rab3GAP activity by thin-layer chromatography. [α- 32 P] GTP-Rab3A was incubated for 5 minutes at 30 ° C. in the presence or absence of a monoQ sample of RAb3GAP (50 ng) and then filtered. Guanine nucleotides bound to Rab3A were eluted from the filter, and the eluted nucleotides were separated by thin-layer chromatography.
【図３】Aは、Rab3GAPに対する基質の脂質修飾の必要性
について検討した結果を示す図である。脂質修飾及び脂
質非修飾のRab3Aに結合した[γ-32P]GTPの加水分解を様
々な量のRab3GAPのモノQサンプルの存在下で測定した。
(●)は脂質修飾したRab3Aを示し、(○)は脂質非修飾のR
ab3Aを示す。Bは、Rab3Aの基質特異性を検討した結果を
示す図である。Rabファミリーの脂質修飾体に結合した
[γ-32P]GTPの加水分解をさまざまな量のモノQサンプル
の存在下で測定した。(●)はRab3Aを、(▲)はRab3Bを、
(■)はRab3Cを、(◆)はRab3Dを、(◇)はRab2を、(□)は
Rab5Aを、(○)はRab11をそれぞれ示す。Cは組換えRab3G
APのGAP活性を検出した結果を示す図である。脂質修飾
されたRab3Aに結合した[γ-32P]GTPをモノQサンプルま
たはRab3GAPの組換えサンプルの存在下で測定した。
(●)はHis6-GAP、(○)はRab3GAPのモノQサンプルを示
す。FIG. 3A is a diagram showing the results of an investigation on the need for lipid modification of a substrate for Rab3GAP. Hydrolysis of [γ- 32 P] GTP bound to lipid-modified and unlipid-modified Rab3A was measured in the presence of varying amounts of Rab3GAP mono-Q samples.
(●) indicates lipid-modified Rab3A, and ()) indicates lipid-unmodified Rab3A.
ab3A is shown. B is a diagram showing the result of examining the substrate specificity of Rab3A. Bound to modified Rab family lipids
[γ- 32 P] GTP hydrolysis was measured in the presence of various amounts of Mono-Q sample. (●) Rab3A, (▲) Rab3B,
(■) Rab3C, (◆) Rab3D, (◇) Rab2, (□)
Rab5A and (○) indicate Rab11, respectively. C is recombinant Rab3G
FIG. 3 is a view showing the results of detecting GAP activity of AP. [Γ- 32 P] GTP bound to lipid-modified Rab3A was measured in the presence of a mono-Q sample or a recombinant sample of Rab3GAP.
(●) shows the His6-GAP, and ()) shows the Rab3GAP mono-Q sample.
【図４】Rab3GAPの推定されたアミノ酸配列を示す図で
ある。Rab3GAPのモノQサンプルから決定されたアミノ酸
配列を下線部に示した。なお、アミノ酸の表記は生化学
事典第2版（東京化学同人）p1468記載の一文字表記に従
った。FIG. 4 shows the deduced amino acid sequence of Rab3GAP. The amino acid sequence determined from the mono-Q sample of Rab3GAP is underlined. In addition, the description of the amino acid followed the one-letter description of the biochemical encyclopedia 2nd edition (Tokyo Kagaku Dojin) p1468.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ (Ｃ１２Ｎ 1/21 Ｃ１２Ｒ 1:19） (Ｃ１２Ｎ 9/14 Ｃ１２Ｒ 1:19） ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification symbol FI (C12N 1/21 C12R 1:19) (C12N 9/14 C12R 1:19)
Claims (8)
バーに結合したGTPの加水分解を特異的に促進する活性
を有するタンパク質。1. A protein having an activity of specifically promoting the hydrolysis of GTP bound to a lipid-modified Rab3 subfamily member.
パク質。2. The protein according to claim 1, which is derived from a mammal.
は該タンパク質中のアミノ酸配列において１若しくは数
個のアミノ酸が置換、欠失、若しくは付加したアミノ酸
配列を有し、脂質修飾されたRab3サブファミリーメンバ
ーに結合したGTPの加水分解を特異的に促進する活性を
有するタンパク質。3. A lipid-modified Rab3 subfamily having the protein of SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence. A protein having an activity of specifically promoting the hydrolysis of GTP bound to a member.
イズするDNAがコードするタンパク質であって、脂質修
飾されたRab3サブファミリーメンバーに結合したGTPの
加水分解を特異的に促進する活性を有するタンパク質。4. A protein encoded by a DNA that hybridizes with the DNA of SEQ ID NO: 2, which has an activity of specifically promoting the hydrolysis of GTP bound to a lipid-modified Rab3 subfamily member. protein.
ドするDNA。5. A DNA encoding the protein according to claim 1.
質転換体。A transformant carrying the vector according to claim 6.
される組み換えタンパク質。8. A recombinant protein produced by the transformant according to claim 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9090706A JPH10276783A (en) | 1997-04-09 | 1997-04-09 | Protein for accelerating hydrolysis of gtp |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9090706A JPH10276783A (en) | 1997-04-09 | 1997-04-09 | Protein for accelerating hydrolysis of gtp |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH10276783A true JPH10276783A (en) | 1998-10-20 |
Family
ID=14005980
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9090706A Pending JPH10276783A (en) | 1997-04-09 | 1997-04-09 | Protein for accelerating hydrolysis of gtp |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH10276783A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7153831B2 (en) | 2002-11-01 | 2006-12-26 | Eisai Co., Ltd. | Rabconnectin-3-binding protein |
-
1997
- 1997-04-09 JP JP9090706A patent/JPH10276783A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7153831B2 (en) | 2002-11-01 | 2006-12-26 | Eisai Co., Ltd. | Rabconnectin-3-binding protein |
| US7279461B2 (en) | 2002-11-01 | 2007-10-09 | Eisai R&D Management Co., Ltd | Rabconnectin-3-binding protein |
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