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1990 Fiscal Year Final Research Report Summary

Molecular Cloning of Human cDNA Encoding a Mannosyltransferase Activity Involved in the Synthesis of the Lipid Intermediate.

Research Project

Project/Area Number 01560109
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 応用生物化学・栄養化学
Research InstitutionThe Tokyo Metropolitan Institute of Medical Science

Principal Investigator

NISHIKAWA Yoshihisa  Senior Researcher, Tumor Biology Section, The Tokyo Metropolitan Institute of Medical Science, 腫瘍細胞研究部門, 研究員 (20109953)

Co-Investigator(Kenkyū-buntansha) YAMAMOTO Yukio  Researcher, Tumor Biology Section, The Tokyo Metropolitan Institute of Medical S, 腫瘍細胞研究部門, 研究員 (80124501)
Project Period (FY) 1989 – 1990
KeywordsOligosaccharideーlipid / Mutant / Cell growth / GRP78 / Bip / Glycosylation / cDNA / Glycosyltransferase / Oligosaccharyltransferase
Research Abstract

I have isolated a temperatureーsensitive (ts) Asnーlinked glycosylation mutant, designated G258, from a mouse mammary carcinoma cell line FM3A by [2-^3H] mannose suicide selection. G258 is not only ts for Asnーlinked glycosylation, but also ts for cell growth. The biochemical defect resides in the formation of oligosaccharidelipid (OL). At 33^゚C, G258 cells synthesize Glc_3Man_9GlcNAc_2-PP-Dol, the fully assembled OL, but at 39^゚C, G258 cells are able to synthesize merely the mediumーsized OL (up to Man_3GlcNAc_2-PP-Dol), but are unable to synthesize the larger OL [J. Cell. Physiol. 119 : 260-266 (1984) ]. Judging from the current model of the topography of OL synthesis in the rER membrane, medium-sized OLs such as Man_3GlcNAc_2-PP-Dol would not be recognized by oligosaccharyltransferase. Consequently, at 39゚C, VSV-infected G258 cells could not perform Asn-linked glycosylation of G (glyco) protein (submitted paper). Moreover, syntheses of GRP78 (Bip) and GRP94 were induced at 39^゚C in G258 … More cells (submitted paper). The GRP78 (Bip) is known to associate with malfolded proteins. This result implies that aberrant foldings and aberrant assemblies of (glyco) proteins occur at 39゚C in G258 cells.
Spontaneous revertants for cell growth were isolated from G258 cells. All growth-revertants also showed reversions on the Asn-linked glycosylation and on the synthesis of OL [Biochim. Biophys. Acta 1091 : 135-140 (1991) ]. These results imply that both temperature-sensitivities of G258 cells were derived from a single gene mutation. This characteristic will enable me to clone human cDNA on the OL synthesis by screening its activity to let G258 cells recover from their temperature sensitivity for cell growth.
A possible mechanism of the coupling between impaired OL synthesis and growth arrest in G258 cells at 39^゚C is as follows : At 39^゚C, G258 cells are unable to synthesize OLs beyond Man_3GlcNAc_2-PP-Dol. Consequently G258 cells are defective in Asn-linked glycosylation. Resultant aglycoproteins fold abnormally, and accumulate in the rER, forming complex with GRP78 (Bip). Thus the functional expression of essential (glyco) proteins for cell growth [perhaps receptors for growth factors, and/or receptors for nutrients] may be impaired. This may lead G258 cells to stop their growth at 39^゚C. Less

  • Research Products

    (24 results)

All Other

All Publications (24 results)

  • [Publications] 西川 義尚: "小胞体膜におけるリピド中間体生合成のトポグラフィ-" 化学と工業. 42. 906-908 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 西川 義尚: "細胞増殖因子レセプタ-の機能発現におけるアスパラギン結合型糖鎖のかかわり" バイオサイエンスとバイオインダストリ-. 47. 930-935 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 西川 義尚: "糖タンパクの細胞内輸送に於けるアスパラギン結合型糖鎖の機能" 日本農芸化学会誌. 64. 930-935 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nishikawa,Y.: "An FM3A mutant, G258, with a mutation that affects both cell growth and oligosaccharideーlipid synthesis." Biochimica et Biophysica Acta. 1091. 135-140 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sarker,M.: "Molecular cloning and expression of cDNA encoding the enzyme that controls conversion of highーmannose to hybrid and complex Nーglycans: UDPーNーacetylglucosamine:αー3ーDーmannoside βー1,2ーNーacetylglucosaminyltransferase I." Proceedings of the National Academy of Sciences,U.S.A.88. 234-238 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 西川 義尚: "[^3H]マンノ-スを用いたトリチウム自殺法による糖鎖生合成変異株の濃縮" 蛋白質・核酸・酵素 増刊号「分子細胞遺伝学実験法」. (1991)

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      「研究成果報告書概要(和文)」より
  • [Publications] 西川 義尚: "糖蛋白のアスパラギン結合型糖鎖生合成に関する変異株" 蛋白質・核酸・酵素 増刊号「分子細胞遺伝学実験法」. (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nishikawa,Y.: "Induction of glucoseーregulated proteins at nonpermissive temperature in a mouse temperatureーsensitive mutant G258, defective in oligosaccharideーlipid synthesis."

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 西川 義尚: "蛋白のアスパラギン残基へのオリゴ糖転移" BIOmedica. (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 西川 義尚、鈴木 旺、山科 郁男、大沢 利昭、木幡 陽、永井 克孝編: "日本生化学会編「新生化学実験講座」第3巻「糖質」I.糖タンパク質(下),§11.1.3.「Nーアセチルグルコサミンの導入」" 東京化学同人, 861 (1990)

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      「研究成果報告書概要(和文)」より
  • [Publications] 西川 義尚、鈴木 旺、山科 郁男、大沢 利昭、木幡 陽、永井 克孝編: "日本生化学会編「新生化学実験講座」第3巻「糖質」I.糖タンパク質(下),§11.1.6.「オリゴ糖転移」" 東京化学同人, 861 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 西川 義尚,(市川 厚,川嵜 敏祐,他編): "「生物薬科学実験講座」第1部「生体構成成分と高分子」4.「糖質」ー第2巻「糖タンパク質実験法」 6ー2「ドリコ-ル中間体の生合成」" 広川書店,

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y. Nishikawa: "Topography of lipid intermediate synthesis in rER membrane." Kagaku to Kogyo. 42. 906-906 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y. Nishikawa: "Role of asparagineーlinked oligosaccharide in the functional expression of growth factor receptor." Bioscience and Bioindustry. 47. 930-935 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y. Nishikawa: "Role of asparagineーlinked oligooligosaccharide in the intracellular transport of glycoprotein." Nippon Nogeikagaku Kaishi. 64. 930-935 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y. Nishikawa: "An FM3A mutant, G258, with a mutation that affects both cell growth and oligosaccharideーlipid synthesis." Biochim. Biophys. Acta. 1091. 135-140 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sarker, M., Hull, E., Nishikawa, Y., Simpson, R. J., Moritz, R. L., Dunn, R. and Schachter, H.: "Molecular cloning and expression of cDNA encoding the enzyme that controls conversion of highーmannose to hybrid and complex N-glycans : UDP-N-acetylglucosamine : -3-D-mannoside -1, 2- Nーacetylglucosaminyltransferase I." Proc. Natl. Acad. Sci. USA. 88. 234-238 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nishikawa, Y.: "Selection of glycosylation mutant by tritiated mannose suicide method." Tanpakushitsu, Kakusan and Koso. (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nishikawa, Y.: "Mutants on asparagineーlinked glycosylation of glycoproteins." Tanpakushitsu, Kakusan and Koso. (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nishikawa, Y.: "Induction of glucoseーregulated proteins at non-permissive temperature in a mouse temperatureーsensitive mutant G258, defective in oligosaccharideーlipid synthesis."

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nishikawa, Y.: "Oligosaccharide transfer to asparagine residues of proteins." Biomedical.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nishikawa, Y.: "Transfer of N-acetylglucosamine." "Shinseikagaku Jikken Koza". vol. 3. 458-474 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nishikawa, Y.: "Oligosaccharide transfer." "Shinseikagaku Jikken Koza". vol. 3. 485-496 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nishikawa, Y.: "Synthesis of dolichol intermediate" "Seibutsu Yakukagaku Jikken Koza". Hirokawa Shoten.

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      「研究成果報告書概要(欧文)」より

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Published: 1993-08-12  

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