JP3889863B2 - Glycosyltransferase gene - Google Patents

Description

配列番号：2
配列の長さ：1613
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：ＤＮＡ

【図面の簡単な説明】
【図１】図１は、β1,3Gal-Tの抗GD1bmAbを用いたcDNAクローニングを示す図であり、KF4CはpolyomaT抗原とβ1,4GalNAc-T遺伝子を導入されており、α2,8シアル酸転移酵素とβ1,3Gal転移酵素のcDNAが共に発現すると、GD1bが発現し得ることを示す図である。
【図２】図２は、B78-2及びL1-17において新規ガングリオシドのトランジエント発現を示す図である。クローン化されたcDNA pM1T-9は、GD3シンターゼcDNA、pD3T-31あり（右）またはなし（左）の条件でDEAE-デキストラン法により遺伝子導入した。GM1,GD1bおよびGA1は60時間後、フローサイトメトリーにより特異的プローブを用いて分析した。細線は特異的プローブありの条件での結果を示し、実線はコントロールを示す。
【図３】図３は、安定遺伝子導入細胞でのGM1/GD1bの発現を示す図である。B78-2細胞は、pM1T-9のみ（左）またはpM1T-9とpD3T-31（右）により遺伝子導入された。GM1はＡおよびＢともに発現し、一方GD1bはＤにのみ発現した。細線はmAbs添加サンプルを、また実線は二次抗体のみで処理したものを示す。
【図４】図４は、安定遺伝子導入細胞から抽出されたガングリオシドのＴＬＣの結果を示す図である。
Ａ：レーン１及び２はB78から、レーン３はB78-2、レーン４はB78-2/M1T-9/D3T-31からの抽出したものである。レーン１は、10mgのウエット組織から、レーン２〜４は5mgのウエット組織からのガングリオシドである。標準は、ウシ脳からのガングリオシドである。使用溶媒は、クロロホルム/メタノール/2.5N NH₄Cl(60:35:8)である。レゾルシノール噴霧をバンド検出に使用する。
Ｂ：ガングリオシドフラクションのTLC-免疫染色。レーン３および４はＡと同様に調製した後、PVDF膜にブロットし、抗GD1b mAb370により染色した（発色にはABCキットを用いた）。
【図５】図５は、クローン化β1,3Gal-T pM1T-9の核酸配列を示す図である。
上：単一のオープンリーディングフレームに対する演繹されたアミノ酸配列を塩基配列に加えて示す図である。２１アミノ酸を有する推定トランスメンブラン領域は二重線で示す。N-グリコシル化の可能性部位が下線で示される。ポリアデニル化シグナルが四角でかこまれる。
下：コード領域の演繹されたアミノ酸配列に対する、Kyte、Doolittle(Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132)に基づくハイドロパシー分析を示す図である。
【図６】図６は、cDNAの安定遺伝子導入体からの抽出物のβ1,3Gal-T活性を示す図である。ここでＡは、GM2アクセプターを用いた場合の酵素活性（UPD-［¹⁴C］-Galactoseをドナーとして用いる）を示す 。メンブランフラクションが親株B78-2とB78-2/M1T-9/D3T-31から調製され、酵素活性がGM2をアクセプターとして用いて決定された。GM1バンドが生成物のTLCで観測された。Ｂは、種々のアクセプター構造に対する相対的β1,3Gal-T活性。GM2アクセプターの場合を100%とした。
【図７】図７は、β1,3Gal-T遺伝子のノーザンブロットの結果を示す写真である。Ａは、ラット各組織からの全RNA20μgをアガロースゲルで分離し、ナイロンメンブレンにブロットする。pM1T-9から調整した³²Ｐ-標識プローブとのハイブリダイズがなされたものである。Ｂは、全ラット胎仔（レーン１）、ラット胎仔脳（レーン２〜３）、またはラット脳（レーン４）のRNAが分離され、Ａと同様にブロットされたものである。レーン５および６は脳からのRNA30μgを用いたものである。
【図８】図８は、図７で得られたバンド強度を、エチジウムブロミドで染色された18S rRNAバンド（デンシトグラフ（Signal Analysis, Vienna, VA)にて測定）を用いて補正し、プロットした図である。繰返し実験の結果を+-SDで表現した。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a glycosyltransferase and a gene encoding the same.
[0002]
[Prior art]
Gangliosides are amphiphilic glycolipid molecules containing sialic acid and are found abundantly in vertebrate nerve tissues (Suzuki, K. (1965) J. Neurochem. 12,969-979). A lot of research has already been done on gangliosides and they play an important role in the development, maintenance and repair of the nervous system (Schaal, H., Wille, C., and Wille, W. (1985) J. Neurochem. 45,544-551), the biological function of gangliosides is neurotrophic factor (Ferrari, G., Anderson, BL, Stephens, RM, Kaplan, DR, and Greene, LA (1995) J. Biol. Chem. 270,3074-3080), receptors for neurotrophic factors and receptors for growth factors. Bremer, EG, Hakomori, S., Bowen-Pope, DF, Raines, E., and ROSS, R. (1984) ) J. Biol. Chem. 259, 6818-6825). Furthermore, it is also known that gangliosides administered systemically into the body exhibit neurotrophic effects in various pathological conditions.
[0003]
On the other hand, the molecular mechanisms by which gangliosides affect nervous system functions and control cell growth and differentiation are still unclear. Conventionally, there has been only a method of exogenously adding ganglioside to cultured cells or experimental animals and observing the effect. These problems are due to the isolation of several glycosyltransferase genes (Joziasse, DH, Shaper, JH, Van den Eijnden, DH, VanTunen, AJ, and Shaper, NL (1989) J. Biol. Chem. 264, 14290- 14297, Narimatsu, H., Sinha, S., Brew, K., Okayama, H., and Qasba, PK (1986) Proc. Natl. Acad. Sci. USA 83, 4720-4724). Has made it possible to modify the type of glycation of cells, which has been resolved (Ikematsu, S., Kaname, T., Ozawa, M., Yonezawa, S., Sato, E., Uehara, F. , Obama, H., Yamamura. K., and Muramatsu, T. (1993) Glycobiology. 3, 575-580, Ioffe, E., and Stanley, P. (1994) Proc. Natl. Acad. Sci. USA 91 , 728-732, Metzler, M., Gertz, A., Sarkar, M., Schachter, H., Schrader, JW, and Marth, JD (1994) EMBO J. 13, 2056-2065). For example, attempts have been made to remodel carbohydrates in cultured cells and genetically modify carbohydrates in experimental animals (Takamiya, K., Yamamoto, A-, Furukawa, K., Yamashiro, S., Shin, M., Okada, M., Fukumoto, S., Haraguchi, M., Takeda, N., Fujimura, K., Sakae, M., Kishikawa, M., Shiku, H., Furukawa, K., And Aizawa, S. (1996) Proc. Natl. Acad. Sci. USA 93,10662-10667), a novel function of carbohydrates that was not expected or not clearly known was found.
[0004]
Of the complex gangliosides, GM1 is the most studied and is the main ganglioside present in the vertebrate brain. GM1 also binds specifically to the cholera toxin B subunit and cAMP response (Holmgren, J., Lonnroth, I., Mansson, JE, and Svennerholm, L. (1975) Proc. Natl. Acad. Sci. USA 72, 2520-2524) are known to bring about important biological phenomena. Furthermore, GM1 is also known to be involved in the receptor function of peptide hormones such as FSH and LH, and to be effective in therapeutic applications for many pathological situations of the nervous system.
[0005]
However, a detailed analysis of the physiological functions of complex gangliosides has caused the problem that the glycosyltransferase genes involved in the synthesis of individual structures are not yet available.
[0006]
[Problems to be solved by the invention]
The present inventors have made extensive studies in view of the above problems, and succeeded in making the glycosyltransferase genes involved in the synthesis of individual structures available for detailed analysis of the physiological functions of complex gangliosides. Is. That is, the present inventors isolated β1,3-galactosyltransferase (EC 2.4.1.-) (β1,3Gal-T) gene whose cDNA clone determines the expression of GD1b. succeeded in. Further, by introducing the cDNA into various cell lines having an appropriate precursor structure, and from an in vitro assay using an extract from a cell into which the cDNA gene has been introduced, the enzyme is converted into GM1 and asialo- It was found to catalyze the synthesis of GM1 (GA1). In addition, important findings were obtained regarding the expression pattern of the enzyme between rat tissues and in the rat brain, and the present invention was completed based on such findings.
[0007]
[Means for Solving the Problems]
That is, in the present invention, cDNA cloning of β1,3-galactosyltransferase gene (EC 2.4.1.62) is performed using an anti-GD1b monoclonal antibody. For this reason, KF4C, a mouse melanoma strain B16, which has been transfected with a polyoma T antigen gene and GM2 / GD2 synthase cDNA, is used as a recipient cell line for cDNA library gene introduction. In addition, pD3T-31, a cDNA clone of GD3 synthase, is co-transfected (co-transfected, co-transfection) with a cDNA library prepared using rat brain RNA and pcDNAI expression vector.
[0008]
Furthermore, according to the present invention, the isolated cDNA clone pM1T-9 has a gene product comprising 4 amino acids in the cytoplasmic domain, 21 amino acids in the transmembrane domain, and 346 amino acids in the large catalytic domain. This suggests that it is a type II membrane protein.
[0009]
In the present invention, the cDNA is introduced into the mouse melanoma strain B16 into which the GM2 / GD2 synthase gene has been introduced, resulting in neo-synthesis of GM1.
[0010]
Furthermore, in the present invention, it was found that when pM1T-9 and GD3 synthase cDNA were co-transfected into the cell line, GD1b was also expressed like GM1. Furthermore, it has been found that introduction of pM1T-9 into an L cell line (GM3 synthase deletion) into which the GM2 / GD2 synthase gene has been introduced in advance expresses asialo-GM1 (asialo-GM1). The findings in the present invention confirm that the GD1b / GM1 / GA1 synthase is the same as suggested by the enzyme analysis.
[0011]
Furthermore, in the present invention, the discovery that 1.6 kb mRNA is strongly expressed in the spleen, thoracic cord, kidney, and testis in the Northern blot of β1,3-galactosyltransferase gene using total RNA from rat tissues. Got. Furthermore, the expression level of the gene in the brain tissue of adult rats is not particularly high, whereas the gene is high in the rat fetal brain on day 12 of the embryonic day, reaches a peak with birth, and then is similar to the brain of adult rats The knowledge that it falls to the low level of.
[0012]
That is, the present invention relates to an amino acid sequence from Ser at position 26 to Ser at position 371 in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing, or one or more, usually 1 to several amino acids relative to the amino acid sequence The present invention provides a glycosyltransferase having β1,3-galactosyltransferase activity, comprising an amino acid sequence deleted, substituted, or added.
[0013]
The present invention also relates to an amino acid sequence from Met at position 1 to Ser at position 371 of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, or one or more, usually 1 to several amino acids relative to the amino acid sequence. The present invention provides a glycosyltransferase having β1,3-galactosyltransferase activity, comprising an amino acid sequence deleted, substituted, or added.
[0014]
Furthermore, the present invention provides a polynucleotide encoding the glycosyltransferase described above.
[0015]
The present invention also provides a polynucleotide having a base sequence from T at position 270 to T at position 1307 among the base sequences set forth in SEQ ID NO: 2 in the sequence listing.
[0016]
The present invention also provides a polynucleotide having a base sequence from A at position 195 to T at position 1307 among the base sequences set forth in SEQ ID NO: 2 in the sequence listing.
[0017]
Furthermore, the present invention provides a polynucleotide that hybridizes with the nucleotide sequence set forth in SEQ ID NO: 2 in the Sequence Listing and encodes a protein (glycosyltransferase) having β1,3-galactosyltransferase activity. Such hybridizing DNA or RNA preferably retains β1,3-galactosyltransferase activity, and under stringent conditions such as 50% formamide, 5 × SSC, 10% Na-dextran, 20 mM Na -Phosphate (pH 6.5), which hybridizes with the polynucleotide under hybridization conditions of 42 ° C.
[0018]
Hereinafter, the present invention will be described in more detail with reference to embodiments. In the present specification and drawings, when abbreviations such as bases and amino acids are used, abbreviations by IUPAC-IUB or abbreviations commonly used in the field are used.
[0019]
(Nucleic acid)
DNA deoxyribonucleic acid
cDNA Complementary DNA
RNA Ribonucleic acid
mRNA Messenger RNA
A adenine
C cytosine
G Guanine
T thymine
(Other)
TLC thin layer chromatography
Trf transfection or transfectant
β-1,4 GalNAc-T β1,4-N-acetylgalactosaminyltransferase
β-1,3 Gal-T β1,3-galactosyltransferase
α2,8S-T α2,8 sialyltransferase
CMP-NeuAc cytocine monophosphate-N-acetylneuraminic acid
UDP-GalNAc uridine diphosphate-N-acetylgalactosamine
UDP-Gal uridine diphosphate galactose
CT-B cholera toxin B
Et.Br. Ethydium bromide
[0020]
DETAILED DESCRIPTION OF THE INVENTION
Materials and methods
(Cells and antibodies)
The mouse cell line KF4C is KF3027 (Nagata, Y., Yamashiro, S., Yodoi, J., Lloyd, KO, Shiku, H., and Furukawa, K. (1992) J. Biol. Chem. 267, 12082- 12089) plasmid pMIK-Hyg / M2T1-1 (pMIK / Hyg vector inserted with GM2 / GD2 synthase cDNA). B78-2 is a stable gene transferer of pM2T1-1 into B78 (B16 subline) (Nagata, Y., Yamashiro, S., Yodoi, J., Lloyd, KO, Shiku, H., and Furukawa, K. (1992) J. Biol. Chem. 267, 12082-12089). These cells consist of Dulbecco modified Eagle's minimum required medium (D-MEM) 7.5% fetal calf serum (FCS) and 300 μg / ml with or without hygromycin (250 μg / ml) (Calbiochem-Novabiochem, La Jolla, Calif.). Can be retained in ml G418 (Sigma, St. Louis, Mo.).
[0021]
L1-17 is a gene-transferred L cell of the GM2 / GD2 synthase cDNA clone. That is, it is incorporated into pCDM8 (Nagata, Y., Yamashiro, S., Yodoi, J., Lloyd, KO, Shiku, H., and Furukawa, K. (1992) J. Biol. Chem. 267, 12082-12089). PM2T1-1 and pSV2neo gene transferer, and asialo-GM2 expression. A monoclonal antibody (mAb370) that specifically reacts with GD1b and an anti-asialo-GM1 monoclonal antibody (mAb229) can be prepared by the present inventors as follows.
[0022]
That is, each ganglioside is incorporated into a liposome and AKR mice are immunized at 2-week intervals, and then the spleen cells are fused with NS-1 myeloma cells. After selecting with HAT medium, ELISA for ganglioside is performed using the culture supernatant and a specific antibody is picked up.
[0023]
(Plasmid and cDNA library)
The rat brain cDNA library is poly (A)⁺It can be prepared using RNA and a pcDNAI expression vector (Invitrogen, San Diego, CA). This library is 3.5x10⁶Those containing one or more independent colonies are preferred.
[0024]
E.Coli MC1061 / P3 can be used as a host bacterium.
[0025]
Plasmid pMIK / D3T-31 is a GD3 synthase cDNA clone, pD3T-31 (Haraguchi, M., Yamashiro, S., Yamamoto, A., Furukawa, K., Takamiya, K., Lloyd, k. 0., XhoI fragment of Shiku, H., and Furukawa, K. (1994) Proc. Natl. Acad. Sci. USA 91, 10455-10459) provided by pMIK / Neo expression vector (Tokyo Medical and Dental University, Dr. Maruyama) It can be prepared by inserting into the cell engineering experiment protocol, “construction of expression plasmid”, pages 254-259, edited by Institute of Medical Science, University of Tokyo, Shujunsha, 1993).
[0026]
(Isolation of β1,3-galactosyltransferase cDNA clone)
The plasmids in the cDNA library were grown once according to conventional methods (Davis, LG, Dibner, MD, and Battey, JF (1986) Methods in Molecular Biology, Elsevier Science. 285-289) and together with the plasmid pMIK / D3T-31. It is possible to introduce a gene into KF4C cells using DEAE dextran (Pharmacia Biotech, Uppsala).
[0027]
For example, a 10cm dish (Corning, Corning, NY) subconfluent KF4C cells 1.5x10⁶This is possible by co-gene transfer of 8 μg each of cDNA library plasmid and pMIK / D3T-31. Further, after 60 minutes, the cells are treated with 10% DMSO / PBS for 2 minutes and then cultured for 2 days. The cells into which the gene has been introduced are detached from the plate, and incubated with mAb 370 at a dilution of 1: 200 for 45 minutes on ice. Cells are transferred to dishes coated with goat anti-mouse IgM (Cappel, Durham, NC) (Wysocki, L. J., and Sato, V. L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2844-2848). Plasmid DNA can be extracted from panned cells by preparing a Hirt extract, and can be transformed into MC1061 / P3. The expanded plasmid DNA is reintroduced and the same operation is repeated (for example, 4 times). Thereafter, 96 groups each containing 30 colonies can be prepared and screened by expressing mAb370 binding activity. An appropriate number (eg 17) of clones from several positive groups (eg 2) was screened and single colonies (eg 3) expressing GD1b on KF4C were selected for microscale DEAE dextran gene transfer and immunofluorescence (IF ) It can be isolated using an assay.
[0028]
(DNA sequence)
The isolated cDNA plasmid can be digested with XhoI and HindIII and cloned into the phagemid bluescript (pBSK) KS (+) vector. A deletion mutant of this clone can be prepared by, for example, the Kilo-Sequence deletion kit (Takara, Kyoto).
[0029]
The dideoxynucleotide termination sequence was determined using a model 377 DNA sequencer (Applied Biosystems, Foster City, Calif.) Using T3 / T7 dye primers or four additional custom primers and PRISM dye terminator cycle sequence kit. Is possible.
[0030]
(Β1,3Gal-T gene)
Mouse melanoma cell lines, KF-4C and GM2-expressing cell lines 78-2 can be used as recipient cells for transient and stable expression systems.
[0031]
For transient expression of GM1, the pM1T-9 / cDNAI vector is introduced into KF-4C by DEAE-dextran.
[0032]
For transient expression of GD1b, pMIK-neo / D3T-31 and pM1T-9 / cDNAI are co-transfected into KF-4C.
[0033]
For transient expression of GA1, pM1T-9 / cDNAI is introduced into GA2-expressing L1-17 cells. The expression of these gangliosides can be detected, for example, by IF assay and flow cytometry.
[0034]
For the preparation of stable GM1 and GD1b expressing gene transfectants, pM1T-9 / cDNAI and pMIK-Hyg / D3T-31 were prepared by, for example, calcium phosphate precipitation (Davis, LG, Dibner, MD, and Battey, JF (1986) in Molecular Biology, Elsevier Science. 290-292). To select the gene transfectant, the cells are cultured in D-MEM / 7.5% FCS containing G418 (300 μg / ml) and hygromycin (250 μg / ml). Expression of gangliosides of these stable gene transfectants is performed by IF assay and flow cytometry.
[0035]
(Flow cytometry analysis)
Ganglioside expression is performed using mouse mAb370 (anti-GD1b), mAb229 (anti-GA1) and FITC-conjugated cholera toxin B subunit (List Biological Laboratories, Cambell, CA) (GM1). Specifically, for mAbs 370 and 229, cells are incubated with mAbs for 45 minutes on ice and stained with FITC-conjugated rabbit anti-mouse IgM for 45 minutes on ice.
[0036]
To analyze GM1 on the cell surface, cells are incubated with FITC-conjugated cholera toxin B subunit for 45 minutes and analyzed by flow cytometry on a FACScan (Becton Dickinson, Mountain View, CA). Specifically, the intensity of staining is measured in arbitrary units as a logarithm of fluorescence intensity, and is displayed on a 4-digit scale.
[0037]
As control cells for flow cytometry, for example, GD1b and GA1 can be adjusted using only secondary antibodies. For GM1 expression, vector-only transgenic cells are used after staining with FITC-conjugated cholera toxin B subunit.
[0038]
(RNA isolation and Northern blot)
RNA can be extracted from adult rat tissue and fetal rat brain using acid phenol (Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159). Specifically, total RNA (20 μg) is separated on a 1.2% agarose-formaldehyde gel and transferred onto a GeneScreenPlus membrane (Dupont, Boston, Mass.). [Α-³²P] -dCTP-labeled rat β1,3Gal-T cDNA (pM1T-9) or β-actin cDNA (nucleotide 69-814) probe was hybridized under known conditions (Nagata, Y., Yamashiro, S., Yodoi, J., Lloyd, KO, Shiku, H., and Furukawa, K. (1992) J. Biol. Chem. 267, 12082-12089) .Analysis can be performed using the BAS2000 Bio-Imaging analyzer (Fuji film, Tokyo ) Etc. are possible.
[0039]
(Glycolipid extraction and thin layer chromatography (TLC) immunostaining)
β1,3Gal-T gene stable gene transfer product is obtained by calcium phosphate precipitation of cloned plasmid into B78-2 melanoma and pMIK-Hyg / D3T-31 (α2,8 sialyltransferase (α2,8S-T) cDNA) Obtained by co-transfection. Among G418 and hygromycin resistant clones, GD1b expression clones can be selected based on the results of IF assay and flow cytometry. Glycolipids can be isolated by conventional methods (Furukawa, K., Clausen, H., Hakomori, S., Sakamoto, J., Look, K., Lundblad, A., Mattes, MJ, and Lloyd, KO (1985) Biochemistry. 24, 7820-7826). Specifically, extraction can be performed sequentially with chloroform / methanol (2: 1, 1: 1, 1: 2) from a packed cell mass of about 300 μl of the transfected cells and a control clone containing only the Hyg gene. After desalting, gangliosides can be separated by DEAE-Sephadex A-50 (Pharmacia LKB) ion exchange chromatography. TLC specifically uses chloroform / methanol / 2.5N NH by high-precision TLC plates (Merck, Darmstdt)._FourCan be done using OH (60: 35: 8). The component can be visualized by spraying resorcinol. Novel synthetic gangliosides can be TLC immunostained on silica gel plates (Merck) on aluminum plates (Furukawa, K., Clausen, H., Hakomori, S., Sakamoto, J., Look, K., Lundblad, A., Mattes, MJ, and Lloyd, KO (1985) Biochemistry. 24, 7820-7826). After TLC, the plate is dried and transferred onto a polyvinylidene difluoride (PVDF) membrane. Plates were incubated for 1 hour in phosphate buffer / saline containing 1% bovine serum albumin to block non-specific adsorption, then incubated with mAbs for 1 hour at room temperature, followed by biotinylated rabbit anti-mouse IgG and avidin Incubate with biotin complex. For this purpose, for example, the Vectastain ABC-PO kit (Vector laboratories, CA) can be used.
[0040]
(Enzyme assay)
β1,3Gal-T can be measured by a known method (Ruan, S., and Lloyd, K. O. (1992) Cancer Res. 52, 5725-5731). Specifically, to prepare the membrane fraction, the sample is dissolved using a nitrogen cavitation apparatus. Nuclei are removed by low speed centrifugation and the supernatant is centrifuged at 105,000g for 1 hour at 4 ° C.
[0041]
To analyze the enzyme activity of β1,3Gal-T, specifically, the reaction mixture contains the following components in 50 μl. 150 mM MnCl₂, 0.375% TritonCF-54 (Sigma), UPD- [¹⁴C] Gal (2.0x10^Fivedpm) (NEN, Boston, MA), and membrane fraction containing 100 μg of protein. After incubating this for 2 hours at 37 ° C., the product was isolated on a C18 Sep-Pack cartridge (Waters, Milford, Mass.) And further TLC and fluorography (Yamashiro, S., Ruan, S., Furukawa, K., Tai, T., Lloyd, KO, Shiku, H., and Furukawa, K. (1993) Cancer Res. 53, 5395-5400).
[0042]
(Homology search)
Nucleotide and amino acid sequence homology searches can be performed using the Internet program BLAST (Natioal Center for Biotechnology Information). The amino acid sequence and hydrophobicity / hydrophilicity analysis can be performed with software GENTYX-MACver 6.1.0 (Software Development, Tokyo).
[0043]
【Example】
(Method of expression cloning of GD1b synthase cDNA)
To isolate a cDNA clone of the GD1b synthase gene, the gene for pM2T1-1, β1,4-N-acetylgalactosaminyltransferase (β1,4-N-acetylgalactosaminyltransferase, β1,4GalNAc-T) cDNA, to KF3027 The transductant was prepared to obtain a new cell line expressing GM2, which was named KF4C. As shown in FIG. 1, after co-transfection of α2, 8S-T cDNA, pD3T-31 and β1, 3Gal-T cDNA contained in the library, GD1b can be expressed.
[0044]
(Isolation of cDNA clone of GD1b synthase gene)
After 5 cycles of gene introduction of the cDNA library, panning with anti-GD1b mAb370 and Hirt extraction, a cDNA clone called pM1T-9 was isolated. After co-transfection of pM1T-9 into pD3T-31 and KF4C, new expression of GD1b was observed as shown in FIG. 2B. In these transient transgenic cells, GM1 was also newly expressed when stained with FITC-conjugated cholera toxin B as shown in FIG. 2A.
[0045]
Thus, it is strongly suggested that GD1b and GM1 synthases are identical and are encoded by a single gene (Iber, H., Kaufmann, R., Pohlentz, G., Schwarzmann, G., and Sandhoff , K. (1989) FEBS Lett. 248, 18-22). For further confirmation, only pM1T-9 was introduced into KF4C, and the expression of GD1b and GM1 was examined. These transgenics expressed GM1 but not GD1b. This is consistent with the expected ganglioside synthesis pathway (Iber, H., Kaufmann, R., Pohlentz, G., Schwarzmann, G., and Sandhoff, K. (1989) FEBS Lett. 248, 18-22). To do. Furthermore, this enzyme has been thought to be identical to GA1 synthase, which catalyzes the synthesis of asialo-GM1 (GA1) from asialo-GM2 (GA2). Therefore, in order to examine the presence or absence of GA1 synthesis by gene introduction of this cDNA, L cells transfected with pM2T1-1 were used as pM1T-9 recipient cells. L cells have GM3 synthase activity (Yamashiro, S., Haraguchi, M., Furukawa, K., Takamiya, K-, yamamoto, A., Nagata, Y., Lloyd, KO, Shiku, H., and Furukawa, K. (1995) J. Biol. Chem. 270, 6149-6155) is missing, so all complex gangliosides are missing. As shown in FIG. 2, gene transfer into L cells by pM2T1-1 (stable) and pM1T-9 (transient) clearly expressed GA1 as detected by GA1-specific mAb229.
[0046]
(Confirmation of β1,3Gal-T activity and its products)
According to the present invention, stable gene-transferred cells of pM1T-9 or pM1T-9 and pMIK / D3T-31 were established using B78-2 (B78 gene-transferred cell line by pM2T1-1). Flow cytometry showed a new and significant level of synthesis of GM1 (FIG. 3B) or GM1 / GD1b (FIG. 3D). Glycosphingolipids extracted from parental cells (B78 / M2T1-1) and double transgenics (B78 / M2T1-1 / M1T-9 / D3T-31) are analyzed by TLC and glycolipids Component conversion was found as expected (FIG. 4). In addition, a specific GD1b band was shown by TLC-immunostaining in the double transgenic cells. This result indicated that the cloned cDNA pM1T-9 was derived from the GD1b / GM1 / GA1 synthase gene.
[0047]
(PM1T-9 insert sequence)
FIG. 5 shows the entire sequence of the pM1T-9 insert determined by constructing pBSK having a HindIII-XhoI fragment between the HindIII and XhoI sites of the clone (see SEQ ID NO: 2 in the Sequence Listing). ). Compared to other glycosyltransferase cDNAs isolated so far, this cDNA is relatively small in size and the total size is 1613 bp. It consists of a 194 bp 5'-untranslated region, a 1113 bp continuous open reading frame, and a 306 bp 3'-untranslated region. There is a single polyadenylation signal and more than 70 polyadenylation regions.
[0048]
The start codon at the beginning of the open reading frame is embedded in a sequence similar to the Kozak common start sequence (Kozak, M. (1986) Cell. 44, 283-292). This open reading frame predicts a protein consisting of 371 amino acids with a molecular weight of 40,976.1 daltons (see SEQ ID NO: 1 in the sequence listing). Recent searches of available protein and nucleic acid databases have not found other genes with significant homology to the cDNA. Four other galactosyltransferase genes, α1, 3Gal-T (Joziasse, DH, Shaper, JH, Van den Eijnden, DH, Van Tunen, AJ, and Shaper, NL (1989) J. Biol. Chem. 264, 14290 -14297), β1,4Gal-T (Narimatsu, H., Sinha, S., Brew, K., Okayama, H., and Qasba, PK (1986) Proc. Natl. Acad. Sci. USA 83, 4720- 4724), GalCer-Gal-T (Schulte, S., and Stoffel, W. (1993) Proc. Natl. Acad. Sci. USA 90, 10265-10269; Stahl, N., Jurevics, H., Morell, P , Suzuki, K., and Popko, B. (1994) J. Neurosci. Res. 38, 234-242) and blood group B synthase α1, 3Gal-T (Yamamoto, F., Marken, J., Tsuji, T., White, T., Clausen, H., and Hakomori, S. (1990) J. Biol. Chem. 265, 1146-1151) show only 10-16% homology in amino acid sequences. Met.
[0049]
Search for the predicted amino acid sequence and hydropathy analysis suggest that this protein has structural features similar to previously known glycosyltransferases. There is a single hydrophobic segment near the amino terminus, which consists of 21 amino acids. This putative signal anchor sequence is thought to place 4 residues in the cytosol and place 346 amino acids in the Golgi lumen as a catalytic region. The predicted amino acid sequence shows that a single N-glycosylation site is present at positions 143-145 and a proline-rich region is present at positions 42-71 (11/30). From the above results, the minimum site required for expression of the enzyme activity is from the 26th amino acid to the terminal 371, excluding the transmembrane region and cytoplasmic region.
[0050]
(The β1,3Gal-TcDNA product transfers galactose to GD2, GM2 and GA2)
Due to the transient and stable gene transfer of pM1T-9, this gene strongly codes for three structures: a single β1,3Gal-T that catalyzes the synthesis of GD1b, GM1 and GA1. It was suggested.
[0051]
The enzyme activity of the membrane fraction of stable transgenic cells was examined. As shown in FIG. 6A, the membrane fraction from the stable gene transduced by BMI pMIK / M1T-9 and pD3T-31 showed GM1 synthase activity of 20,000 units (pmol / h / mg protein), while acceptor was added. If not, no significant product was detected. As expected, the transfectant with the pMIK vector itself was almost zero activity. When examined using GD2, GA2, GlcCer, GD1b, GM3 and GD1a as substrates, GD2 and GA2 showed significant uptake levels of UDP-Gal (FIG. 6B).
[0052]
(Northern blot analysis)
The expression level of β1,3Gal-T gene in various rat tissues was examined by Northern blot using total RNA. Of the various tissues, kidney, testis, spleen and thoracic line showed relatively high levels of expression, while almost all tissues examined contained some level of gene transcript (FIG. 7A).
[0053]
Gene expression in adult rat brain tissue was not high. That is, as a result of analyzing the gene expression at the growth stage of the rat brain, the gene was already detected in the fetal brain on the 12th day and maintained at a high level until birth (FIGS. 7B and 8), but in the adult rat brain The expression level is kept low.
[0054]
【The invention's effect】
The present invention demonstrates the identity of the GD1 / GM1 / GA1 synthase gene. That is, GD1b synthase cDNA was isolated, and its gene product was any of GalNAcβ1-> 4Gal-, GalNAcβ1-> 4 (NeuAcα2-> 3) Gal- or GalNAcβ1-> 4 (NeuAcα2-> 8NeuAcα2-> 3) Gal- It is shown that a glycolipid having the terminal sequence is also used as an acceptor.
[0055]
GM2 / GD2 synthase (Yamashiro, S., Haraguchi, M., Furukawa, K., Takamiya, K-, yamamoto, A., Nagata, Y., Lloyd, KO, Shiku, H., and Furukawa, K. ( 1995) J. Biol. Chem. 270, 6149-6155), the GA2 synthesis is relatively low, but in comparison with this, according to the present invention, β1,3 Gal- It was suggested that the acceptor activity of T to GM2, GD2, and GA2 is not so different (FIG. 6B).
[0056]
Furthermore, according to the present invention, the physiological significance of such gangliosides can be studied more physiologically by genetic modification of their glycosyltransferase genes. In other words, the genetic manipulation of the β1,3Gal-T gene ultimately enables the elucidation of the role of higher-grade complex gangliosides than GD1b / GM1 / GA1.
[0057]
For example, as already mentioned, ganglioside is a molecule that plays an important role in the development of the vertebrate nervous system, and in particular, GM1 has been actively studied for its physiological effects in vivo and in vitro. For example, repair effects on damage caused by toxicants, nerve trauma, ischemic damage, degenerative diseases and mechanical invasion (Sabel, BA, Slavin, MD, and Stein, DG (1984) Science. 225, 340-342, Kapriak, SE (1984) Adv. Exp. Med. Biol. 174, 489-497, Svennerholm, L. (1994) Life Sci. 55, 2125-2134). In addition, addition of GM1 ganglioside in an in vitro culture system can induce or enhance neurite outgrowth of neurons (Facci, L., Leon, A., Toffano, G., Sonnino, S., Ghidoni, R , and Tettamanti, G. (1984) J. Neurochem. 42,299-305). In fact, mice with a disrupted β1,4GalNAc-T gene lacking complex gangliosides have reduced neurotransmission rates (Takamiya, K., Yamamoto, A-, Furukawa, K., Yamashiro, S., Shin, M., Okada, M., Fukumoto, S., Haraguchi, M., Takeda, N., Fujimura, K., Sakae, M., Kishikawa, M., Shiku, H., Furukawa, K., And Aizawa, S. (1996) Proc. Natl. Acad. Sci. USA 93,10662-10667) and behavioral abnormalities are clearly shown. In order to elucidate the physiological functions corresponding to these effects, the high expression of β1,3Gal-T gene found only in the developmental stage of rat brain found in the present invention is a ganglioside product in the differentiation of brain tissue. This suggests an important role for gangliosides and their synthase genes, and enables their application to therapeutic drugs.
[0058]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 371
Sequence type: amino acid
Topology: Linear
Sequence type: Protein

SEQ ID NO: 2
Sequence length: 1613
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: DNA

[Brief description of the drawings]
FIG. 1 is a diagram showing cDNA cloning using β1,3Gal-T anti-GD1bmAb, and KF4C has polyomaT antigen and β1,4GalNAc-T gene introduced, and α2,8 sialic acid transfer. It is a figure which shows that GD1b can be expressed when both an enzyme and cDNA of β1,3Gal transferase are expressed.
FIG. 2 is a diagram showing transient expression of novel gangliosides in B78-2 and L1-17. The cloned cDNA pM1T-9 was transfected by the DEAE-dextran method with or without the GD3 synthase cDNA, pD3T-31 (right) or without (left). GM1, GD1b and GA1 were analyzed after 60 hours using specific probes by flow cytometry. The thin line shows the results with the specific probe, and the solid line shows the control.
FIG. 3 is a diagram showing the expression of GM1 / GD1b in stable gene-introduced cells. B78-2 cells were transfected with pM1T-9 alone (left) or pM1T-9 and pD3T-31 (right). GM1 was expressed in both A and B, while GD1b was expressed only in D. The thin line shows the sample treated with mAbs, and the solid line shows the sample treated with the secondary antibody alone.
FIG. 4 is a diagram showing TLC results of gangliosides extracted from stable gene-transferred cells.
A: Lanes 1 and 2 are extracted from B78, lane 3 is extracted from B78-2, and lane 4 is extracted from B78-2 / M1T-9 / D3T-31. Lane 1 is ganglioside from 10 mg wet tissue and lanes 2-4 are 5 mg wet tissue. The standard is ganglioside from bovine brain. The solvent used is chloroform / methanol / 2.5N NH_FourCl (60: 35: 8). Resorcinol spray is used for band detection.
B: TLC-immunostaining of ganglioside fraction. Lanes 3 and 4 were prepared in the same manner as A, then blotted on a PVDF membrane and stained with anti-GD1b mAb370 (ABC kit was used for color development).
FIG. 5 shows the nucleic acid sequence of cloned β1,3Gal-T pM1T-9.
Top: A figure showing the deduced amino acid sequence for a single open reading frame in addition to the base sequence. The putative transmembrane region with 21 amino acids is indicated by a double line. Potential sites for N-glycosylation are underlined. The polyadenylation signal is squared.
Below: Hydropathy analysis based on Kyte, Doolittle (Kyte, J., and Doolittle, RF (1982) J. Mol. Biol. 157, 105-132) for the deduced amino acid sequence of the coding region. .
FIG. 6 is a graph showing β1,3Gal-T activity of an extract from a cDNA stable gene transductant. Here, A is the enzyme activity (UPD- [¹⁴C] -Galactose as donor). Membrane fractions were prepared from parent strains B78-2 and B78-2 / M1T-9 / D3T-31, and enzyme activity was determined using GM2 as an acceptor. A GM1 band was observed in the product TLC. B is the relative β1,3Gal-T activity for various acceptor structures. The case of GM2 acceptor is 100%.
FIG. 7 is a photograph showing the results of Northern blotting of the β1,3Gal-T gene. A: 20 μg of total RNA from each rat tissue is separated on an agarose gel and blotted onto a nylon membrane. Adjusted from pM1T-9³²Hybridized with a P-labeled probe. In B, RNA of whole rat fetus (lane 1), rat fetal brain (lanes 2 to 3), or rat brain (lane 4) was isolated and blotted in the same manner as A. Lanes 5 and 6 use 30 μg of RNA from the brain.
FIG. 8 is a plot of the band intensities obtained in FIG. 7 corrected using an 18S rRNA band stained with ethidium bromide (measured with a densitograph (Signal Analysis, Vienna, VA)). FIG. The results of repeated experiments are expressed as + -SD.

Claims (5)

  Of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, an amino acid sequence from Ser at position 26 to Ser at position 371, or an amino acid sequence in which one or more amino acids have been deleted, substituted or added to the amino acid sequence A glycosyltransferase having β1,3-galactosyltransferase activity, comprising:   An amino acid sequence from Met at position 1 to Ser at position 371 of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added to the amino acid sequence A glycosyltransferase having β1,3-galactosyltransferase activity, comprising:   A polynucleotide encoding the glycosyltransferase according to claim 1 or 2.   A polynucleotide having a base sequence from T at position 270 to T at position 1307 among the base sequences set forth in SEQ ID NO: 2 in the Sequence Listing.   A polynucleotide having a base sequence from A at position 195 to T at position 1307 among the base sequences set forth in SEQ ID NO: 2 in the Sequence Listing.

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