糖鎖遺伝子による細胞リモデリング

• Project/Area Number: 10178104
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (A)
• Allocation Type: Single-year Grants
• Research Institution: Osaka University
• Principal Investigator: 谷口 直之 大阪大学, 医学系研究科, 教授 (90002188)
• Co-Investigator(Kenkyū-buntansha): 鈴木 明身 理化学研究所, 国際フロンティア, グループリーダー (70134533) ; 神奈木 玲児 大阪大学, 愛知県癌センター, 部長 (80161389) ; 木下 タロウ 大阪大学, 微生物研究所, 教授 (10153165) ; 辻 崇一 (財)基礎腫瘍学研究会付属腫溜研究所, 研究員 (90124677) ; 古川 鋼一 名古屋大学, 医学部, 教授 (80211530)
• Project Period (FY): 1998 – 2000
• Project Status: Completed (Fiscal Year 2000)
• Budget Amount: ¥287,400,000 (Direct Cost: ¥287,400,000); Fiscal Year 2000: ¥100,000,000 (Direct Cost: ¥100,000,000); Fiscal Year 1999: ¥78,000,000 (Direct Cost: ¥78,000,000); Fiscal Year 1998: ¥109,400,000 (Direct Cost: ¥109,400,000)
• Keywords: 糖鎖 / 糖脂質 / 糖タンパク質 / 複合糖質 / 糖鎖リモデリング / 糖鎖遺伝子 / 糖転移酵素

Research Abstract

N-グリカンにbisectingGlcNAcを導入する糖転移酵素、GnT-IIIを導入した細胞ではEGFレセプターのエンドサイトーシスが亢進していること、またEGF刺激によるβ-カテニンのリン酸化が低下していることを見い出した。ドリコールリン酸マンノース合成酵素を制御するDPM2が、GPIアンカー合成の初期酵素をも制御することを見いだし、N型糖鎖とGPIアンカーの合成が連動していることを示した。細胞接着分子L-セレクチンの特異的リガンドと同定したシアリル6-スルホルイスxが、大腸上皮細胞に発現し、細胞の悪性化に伴って陰性化する傾向を有する事を見出した。これと対照的に、細胞が悪性化すると硫酸基を持たないシアリルルイスxの発現が増大する。正常大腸上皮細胞では、シアル酸の環状化によってセレクチンとの結合活性が調節されているが、悪性細胞ではこの調節機構が失われていることも判明した。ガングリオシド糖鎖合成に働く新しいシアル酸転移酵素遺伝子(ST6GalNAcV、VI)、およびグロボ系糖脂質合成に働くGb3合成酵素、Gb4合成酵素遺伝子を単離して機能を明らかにした。また、GM2/GD2合成酵素遺伝子ノックアウト、GD3合成酵素遺伝子ノックアウトマウスを用いて、複合型およびβ系列ガングリオシドが神経系維持と修復に必須なことを明らかにした。マウスシアル酸転移酵素のゲノム構造解析などのデータをまとめた。第3の新規Gal:→3硫酸転移酵素のcDNAクローニングを行い、O-結合型糖鎖のcore1鎖(Galβ1→3GalNAcα1→)に特異的に作用することを明らかにした。さらに基質特異性が既知のものとは異なる第4の酵素であるGal:→3硫酸転移酵素をブタ大腸から精製し、酵素学的性状を明らかにした。糖脂質硫酸転移酵素遺伝子のノックアウトマウスは、神経機能障害と精子形成障害をきたしたことから、硫酸化糖脂質のスルファチドとセミノリピドは、それぞれミエリン機能と精母細胞の分化に必須の分子であることが明らかとなった。

Research Products

• [Publications] Tsuda T., et al.: "The Asn-420-linked sugar chain in human epidermal growth factor receptor suppresses ligand-independent spontaneous oligomerization : Possible role of a specific sugar chain in controllable receptor activation."J.Biol.Chem.. 275. 21988-21994 (2000)
• [Publications] Taguchi T., et al.: "Purification and characterization of UDP-GlcNAc : GlcNAcβ1-6 (GlcNAcβ1-2) Man α 1-R [GlcNAc to Man] β1,4N-acetylglucosaminyltransferase VI from hen oviduct."J.Biol.Chem.. 275. 32598-32602 (2000)
• [Publications] Sakamoto Y., et al.: "Molecular cloning and expression of cDNA encoding chicken UDP-GlcNAc : GlcNAcβ1-6 (GlcNAcβ1-2) Man α 1-R [GlcNAc to Man] β1,4N-acetylglucosaminyltransferase VI."J.Biol.Chem.. 275. 36029-36034 (2000)
• [Publications] Sasai K., et al.: "The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V : The involvment of a domain homophilic interaction."J.Biol.Chem.. 276. 759-765 (2001)
• [Publications] Kitada T., et al.: "The Addition of bisecting N-acetylglucosamine residues to E-cadherin downregulates the tyrosine phosphorylation of beta-catenin."J.Biol.Chem.. 276. 475-480 (2001)
• [Publications] Honke K., et al.: "Molecular Cloning And Characterization of A Human β-Gal 3'-Sulfotransferase Which Acts on Both Type 1 and Type 2 (Gal β,3/1,4GlcNAc-R) Oligosaccharides."J.Biol.Chem.. 276. 267-274 (2001)
• [Publications] Sato Y., et al.: "Overexpression of N-acetylglucosaminyltransferase III enhances the EGF-induced phosphorylation of ERK in HeLaS3 cells by upregulation of the internalization rate of the receptors."J.Biol.Chem.. (in press).
• [Publications] Hong,Y. et al.: "Requirement of PIG-F and PIG-O for transferring phosphoethanolamine to the third mannose in glycosylphosphatidylinositol."J.Biol.Chem.. 275. 20911-20919 (2000)
• [Publications] Watanabe,R. et al.: "Initial enzyme forglycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2."EMBO J.. 19. 4402-4411 (2000)
• [Publications] Maeda,Y. et al.: "PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER."EMBO J.. 20. 250-261 (2001)
• [Publications] Kumamoto,K. et al.: "Increased expression of UDP-galactose transporter mRNA in human colon cancer tissues and its implication in synthesis of Thomsen-Friedenreich antigen and sialyl Lewis A/X determinants."Cancer Res.. (in press).
• [Publications] Izawa,M., et al.: "Expression of sialyl 6-sulfo Lewis x is inversely correlated with conventional sialyl Lewis x expression in human colorectal cancer."Cancer Res.. 60. 1410-1416 (2000)
• [Publications] Fukumoto,S. et al.: "GD3 synthase gene expression in PC12 cells results in the continuous activation of TrkA and ERK1/2 and enhanced proliferation."J.Biol.Chem.. 275. 5832-5838 (2000)
• [Publications] Okajima,T. et al.: "Molecular cloning and expression of mouse GD1a/GT1aa/GQ1ba synthase (ST6GalNAc VI) gene."J.Biol.Chem.. 275. 6717-6723 (2000)
• [Publications] Kojima,Y. et al.: "Molecular cloning of Gb3/CD77 synthase, a glycosyltransferase that initiate the synthesis of globo-series glycosphingolipids."J.Biol.Chem.. 275. 15152-15156 (2000)
• [Publications] Honda,K. et al.: "Degradation of human aurora2 protein kinase by the anaphase-promoting complex-ubiquitin-proteasome pathway."Oncogene. 19. 2812-2819 (2000)
• [Publications] Furukawa,K. et al.: "Molecular basis for the p phenotype : Identification of distinct and multiple mutations in the a1,4-galactosyltransferase gene in Swedisn and Japanese individuals."J.Biol.Chem.. 275. 37752-37756 (2000)
• [Publications] Okajima,T. et al.: "Expression cloning of human globoside synthase cDNAs : Identification of b3Gal-T3 as UDP-N-acetylgalactosamine : globotriaosylceramide b1,3-N-acetylgalactosa-minyltransferase."J.Biol.Chem.. 275. 40498-40503 (2000)
• [Publications] Hitoshi S. et al.: "Dorsal root ganglia neuron-specific promoter activity of the rabbit b-galactoside a1,2-fucosyltransferase gene."J.Biol.Chem.. 274. 389-396 (1999)
• [Publications] Lee Y-C. et al.: "Molecular cloning and functional expression of two members of mouse NeuAca2,3Galb1,3GalNAc GalNAc a2,6-sialyltransferase family, ST6GalNAc III and IV."J.Biol.Chem.. 274. 11958-11967 (1999)
• [Publications] Ko J. H., et al.: "Regulation of the GnT-V promoter by transcription factor Ets-1 in various cancer cell lines"J. Biol. Chem.. 274. 22941-22948 (1999)
• [Publications] Miyoshi E. et al.: "Overexpression of α1-6 fucosyltransferase in hepatoma cells suppresses intrahepatic metastasis after splenic injection in athymic mice"Cancer Res.. 59. 2237-2243 (1999)
• [Publications] Hong Y. et al.: "GPII stabilizes an enzyme essential in the first step of glycosylphosphatidyl inositol biosynthesis"J. Biol. Chem.. 274. 18582-18588 (1999)
• [Publications] Kimura, N. et al.: "Reconstitution of functional L-selectin ligands on a cultured human endothelial cell line by cotransfection of α1, 3 fucosyltransferase VII and newly cloned GlcNAcβ : 6-sulfotransferase cDNA"Proc. Natl. Acad. Sci. USA. 96. 4530-4535 (1999)
• [Publications] Mitsuoka C. et al.: "Regulation of selectin binding activity by cyclization of sialic acid moiety of carbohydrate ligands on human leukocytes"Proc. Natl. Acad. Sci. USA. 96. 1597-1602 (1999)
• [Publications] Bistrup A. et al.: "Sulfotransferases of two specificities function in the reconstitution of high-endothelial-cell ligands for L-selectin"J. Cell. Biol.. 145. 899-910 (1999)
• [Publications] Hitoshi, S. et al.: "Dorsal root ganglia neuron-specific promoter activity of the rabbit beta-galactoside alpha-1, 2-fucosyltransferase gene"J. Biol. Chem.. 274. 389-396 (1999)
• [Publications] Lee, Y. C. et al.: "Molecular cloning and functional expression of two members of mouse NeuAc-alpha-2, 3 Gal-beta-1, 3GalNAc GalNAc allpha-2, 6-sialytransferase family, ST6GalNAc III and IV"J. Biol. Chem.. 274. 11958-11967 (1999)
• [Publications] Okajima T. et al.: "Human homolog of Caenorhabditis elegans sqv-3 gene is galactosyl-transferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans"J. Biol. Chem.. (in press).
• [Publications] Fukumoto S. et al.: "Expression cloning of mousse cDNA of CMP-NeuAc : lactosylceramide alpha2, 3-sialyltransferase (GM3 synthase), an enzyme that intiates the synthesis of gangliosides"J. Biol. Chem.. 274. 9271-8276 (1999)
• [Publications] Okajima T. et al.: "Molecular cloning of a novel alpha2, 3-sialyltransferase (ST3Gal VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids"J. Biol. Chem.. 274. 11479-11486 (1999)
• [Publications] Zhao J. et al.: "Attenuation of the interleukin 2 signals in complex ganglioside-lacking mice"J. Biol. Chem.. 274. 13744-13747 (1999)
• [Publications] Memon R. A. et al.: "Regulation of glycosphingolipid metabolism in liver during the acute phase response"J. Biol. Chem.. (in press).
• [Publications] Komori H. et al.: "Regulation of intracellular ceramide content in B16 melanoma cells : Biological implications of ceramide glycosylation"J. Biol. Chem.. 374. 8981-8987 (1999)
• [Publications] R.Watanabe et al.: "The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A,PIG-H,PIG-C and GPII." EMBOJ.17. 877-885 (1998)
• [Publications] S.Tomita et al.: "A homologue of Saccharomyces cerevisiae Dpmlp is not sufficient for synthesis of dolichol-phosphate-mannose in mammalian cells." J.Biol.Chem.273. 9249-9254 (1998)
• [Publications] Y.Maeda et al.: "DPM2 regulates biosynthesis of dolichol phosphate-mannose in mammalian cells : correct subcellular localization and stabilization of DPMI,and binding of dolichol phosphate." EMBOJ.17. 4920-4929 (1998)
• [Publications] A.Irie et al.: "The molecular basis for the absence of N-glycolylneuraminic acid in humans." J.Biol.Chem.273. 15866-15871 (1998)
• [Publications] S.Hitoshi et al.: "Dorsal root ganglia neuron-specific promoter activity of the rabbit β-galactoside α1,2-fucosyltransferase gene." J.Biol.Chem.274. 389-396 (1999)
• [Publications] S.Takashima et al.: "Genomic structure and promoter activity of the mouse polysialic acid synthase(mST8Sia IV/PST)gene." J.Biol.Chem.273. 7675-7683 (1998)
• [Publications] T.Okajima et al.: "Molecular cloning of a novel α2,3-sialyltransferase(ST3Gal VI)that sialylates type II lactosamine structures on glycoproteins and glycolipids." J.Biol.Chem.(in press).
• [Publications] K.Honke,et al.: "Cancer-associated expression of glycolipid sulfotransferase gene in human renal cell carcinoma cells." Cancer Res.58. 3800-3805 (1998)
• [Publications] K.Fukushima et al.: "Elevation of alfa2-6 sialyltransferase and alfal-2 fucosyltransferase activities in human choriocarcinoma." Cancer Res.58. 4301-4306 (1998)
• [Publications] Y.Ihara et al.: "Ectopic expression of N-acetylglucosaminyltransferase III in transgenic hepatocytes disrupts apolipoprotein B secretion and induces aberrant cellular morphology with lipid atorage." Proc.Natl.Acad.Sci.USA.95. 2526-2530 (1998)
• [Publications] Y.Sakamoto et al.: "Purification and characterization of UDP-GlcNAc : Galβ1-4GlcNAcβ1-3Galβ1-4Glc(NAc)-R(GlcNAc to Gal)β1,6N-acetylglucosaminyltransferase from hog small intestine." J.Biol.Chem.273. 27625-27632 (1998)
• [Publications] E.Miyoshi et al.: "Overexpression of α1-6fucosyltransferase in hepatoma cells suppressed intrahepatic metastasis after splenic injection in athymic mice." Cancer Res.(in press).
• [Publications] N.Kimura et al.: "Reconstitution of funcilonal L-selectin ligands on a cultured human endothelial cell line by co-transfection of α1->3 fucosyltransferase VII and newly cloned GlcNAcβ : 6-sulfotransferase cDNA." Proc.Natl.Acad.Sci.USA. (in press).
• [Publications] M.Nakamura et al.: "Single glycosyltransferase,core 2 β-1,6-N-acetylglucosaminyltransferase,regulates cell surface sialyl-Lex expression level in human pre-B lymphocytle leukemia cell Hne KM3 treated with phorbolester." J.Biol.Chem.273. 26779-26789 (1998)
• [Publications] T.Kudo et al.: "Expression cloning and characterization of a novel murine α1,3-fucosyltransferase,mFuc-TIX,that synthesizes the Lewis x(CD15)epitope in brain and kindney." J.Biol.Chem.273. 26729-26738 (1998)
• [Publications] H.Narimatsu et al.: "Lewis and Secretor gene dosages affect CA19-9 and DU-PAN-2 serum levels in normal individuals and colorectal cancer patients." Cancer Res.58. 512-518 (1998)
• [Publications] K.Takamiya et al.: "Complex gangliosides are essential in spermatogenesis of mice : Possible roles in transport of testosterone." Proc.Natl.Acad.Sci.USA. 95. 12147-12152 (1998)
• [Publications] S.Fukumoto et al.: "Expression cloning of mouse cDNA of CMP-NeuAc : lactosylceramide α2,3-sialyltransferase(GM3 synthase),an enzyme that initiates the synthesis of gangliosides." J.Biol.Chem.(in press).
• [Publications] M.Tanemura et al.: "Reduction of the major swine xenoantigen,the α-galactosyl epitope by transfection of the α2,3-sialyltransferase gene." J.Biol.Chem.273. 16421-16425 (1998)
• [Publications] J.Mitoma et al.: "Occurrence of an unusual lipid,phosphatidyl-L-threone,in cultured hippocampal neurons : exogenous L-serine is requiredfor the synthesis of neuronal phosphatidyl-L-serine and sphingolipids" J.Biol.Chem.273. 19363-19366 (1998)
• [Publications] H.Komori et al.: "Regulation of intracellular ceramide content in B16 melanoma cells : Biological implications of ceramide glycosylation" J.Biol.Chem.(in press).
• [Publications] C.Mitsuoka et al.: "Identification of a major carbohydrate capping group of the L-selectin ligand on high endothelial venules in human lymph nodes as 6-sulfo sialyl Lewis x." J.Biol.Chem.273. 11225-11233 (1998)
• [Publications] K.Uchimura et al.: "Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase." J.Biol.Chem.273. 22577-22583 (1998)
• [Publications] C.Mitsuoka et al.: "Regulation of selectin binding activity by cyclization of sialic acid moiety of carbohydrate ligands on human leukocytes." Proc.Natl.Acad.Sci.USA. 96. 1597-1602 (1999)